Clinical Significance And Assessment of 25 OH Vitamin D

Transcription

1 Clinical Significance And Assessment of 25 OH Vitamin D by Bruce Hollis, PhD; Gordon MacFarlane, PhD; and André Valcour, PhD Presented by qwertyui

2 Clinical Significance And Assessment of 25 OH Vitamin D Learning Objectives By Bruce Hollis, PhD; Gordon MacFarlane, PhD; and André Valcour, PhD 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. Introduction and Overview The discovery of vitamin D and the implementation of measures to reduce the incidence 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 deficiency, was rampant, especially in the cities of industrialized nations throughout the world. Fortification of milk with vitamin D has all but eliminated 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 physical well-being. We also consider the causes of the near epidemic of vitamin D deficiency 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 children and adults, suffer from vitamin D deficiency. 1 This situation has occurred, in part, because the foods of the typical 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 vitamin D. 1-3 Most 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 vitamin D in maintaining bone health began to be understood, many manufacturers started 72 advance /LABORATORY FEBRUARY web.com

3 adding vitamin D to food products. Examples of foods that were fortified with vitamin D included such name brands as Bond Bread, Rickter s Hot Dogs, Twang Soda and even Schlitz Beer. 1 The unregulated supplementation with vitamin D, however, resulted in several cases of overdose; consequently, by the 1950s most manufacturers stopped adding vitamin D to their products. 1 In the United States, vitamin D is still added to milk to prevent the occurrence of rickets in the pediatric population. Unfortunately, too many children do not drink enough milk to raise their vitamin D levels to the optimum range. Also, recent studies have shown that the level of vitamin D in fortified milk is frequently much lower than that recommended 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 supplemental 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 epidemic proportions. 1,4 In the early 1800s, Polish physician Jedrzej Sniadecki observed a much higher incidence of rickets in children who lived in the urban environment of Warsaw compared to children who lived in the country. 1 Dr. Sniadecki found that the condition of the city children with rickets improved dramatically when he took them into the countryside and had them spend time in the sun. He correctly surmised that the relative lack of sun exposure experienced by the city children was the cause of their condition. 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. 1 In 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 exclusively. 5 He also showed that he could cure the rickets if he supplemented the oatmeal with cod-liver oil. In 1937, Windaus and Bock discovered that 7-dehydrocholesterol in the skin is converted 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 controlled and depends primarily on the duration and intensity of the UV irradiation. Levels typically reach a plateau within 30 minutes of exposure. 6 Overproduction of vitamin D in the skin is prevented by the photosensitive conversion of vitamin D to tachysterol or lumisterol. 6 Vitamin D is not very water-soluble, so it must be delivered to and carried in the blood as a complex with vitamin D-binding protein. 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 reservoir in the plasma and is generally the best indicator of overall vitamin D status D is further metabolized by the kidney to produce the biologically active form of vitamin D, 1, 25-dihydroxy 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 homeostasis. 6 Several factors are associated 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 Deficiency 1,2,6 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 surgical resection of stomach or intestines are at risk. Individuals with severe liver disease: Hepatic disease can reduce the conversion of vitamin D to 25-D and can lead to malabsorption of vitamin D. Individuals with kidney disease: Nephrotic syndrome can increase the urinary 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 climates 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. web.com advance /LABORATORY FEBRUARY

4 Table 2 Conditions Associated With Vitamin D Deficiency Osteomalacia 1 Osteoporosis 1 Rickets 1,4 Fibromyalgia 1 Prostate Cancer 1,7 Breast Cancer 1,7 Colon Cancer 1,7 Heart Disease 8 Hypertension 8 Multiple Sclerosis 1 Type I Diabetes 1,9 people with dark skin and the elderly. 1,3 Melanin in the epithelial cells is the compound that gives skin its color. 3 This pigment acts as a natural sunscreen, protecting 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 individuals living in northern latitudes can very easily become vitamin D-deficient. Recent studies have indicated that as many as 60 percent of black adults in the United States are vitamin D deficient. 1 The ability of the skin to produce vitamin D on exposure to the sun decreases fourfold from age 20 to age This diminished vitamin D production efficiency 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 elderly black Americans are vitamin D deficient. 1 Many people do not spend enough time in the sun to allow for the production of adequate amounts of vitamin D. 1 The same factors 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 individuals have become aware of the association between sun exposure and the development of wrinkles as well as melanoma and nonmelanoma 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 calcium homeostasis and in the maintenance of healthy bone ,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. 2 Vitamin D deficiency leads to the mobilization of calcium from bone. 6 While it may appear that bones are inert structural elements, they are, in fact, dynamic organs. Human bones continually undergo a tightly coupled process of degradation mediated by osteoclasts and rebuilding mediated by osteoblasts. Working in concert with parathyroid hormone, 1,25-D supports increased bone resorption by mobilizing calcium and phosphate. 2 1,25-D also stimulates new bone formation by inducing osteoblast cells to synthesize alkaline phosphatase and osteocalcin. 2 In children and adolescents, 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 percent and 2 percent 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 important 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 osteoporosis. Caucasians suffer from a significantly greater incidence of osteoporosis than blacks of African genetic lineage 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 properly. 1 Vitamin D deficiency is the most common cause of osteomalacia. Chronic osteomalacia 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 asymptotic until a fracture occurs, patients with osteomalacia suffer from unrelenting deep bone pain and muscle aches. Because many clinicians fail to consider vitamin D deficiency in their differential 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 actually have vitamin D deficiency-related osteomalacia. 1 Osteomalacia-associated vitamin D deficiency may be playing a contributory role in the increased prevalence of obesity in the United States. 1 Since vitamin D is lipophilic, it tends to be absorbed by the excess fat of overweight patients, resulting 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 factors simultaneously serve to exacerbate the weight gain by diminishing physical activity and reduce the person s exposure to the sun, resulting in more profound vitamin D deficiency. This 74 advance /LABORATORY FEBRUARY web.com

5 cycle contributes to the difficulty 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 actively growing, rickets also causes their bones to form improperly. A classic physical 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-normal 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 clinicians for a number of years. There is now evidence suggesting that sun exposure and vitamin D levels may also play a significant role in the health of other organ systems. The geographic distribution of the incidence rates for several cancers is strikingly similar to the distribution of the intensity of UV irradiation necessary for 25-D production. 8 Diminished average 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 pressure. 8 A similar course of treatment with UVA radiation had no effect on 25-D or blood pressure. Epidemiological studies reveal that individuals living close to the equator have a lower incidence of several autoimmune diseases than people living at higher latitudes. For example, the incidence of multiple sclerosis is approximately 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 winter sun, has the world s highest reported incidence of this disease. 1 When 12,000 Finnish babies were given vitamin D supplements, their likelihood of developing diabetes was decreased 80 percent relative 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 scientists 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. 2 This classic model was developed based on an understanding of the relationship between plasma 1,25-D levels and parathyroid hormone in the maintenance of bone health. Recent studies have found that, contrary to this previous understanding, cells from a large number 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 maturation. 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. 9 This in situ tissue 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 hormonally active 1,25-D. 1,6 Chronic vitamin D deficiency 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 insufficiency, has received considerable attention in recent years. Many investigators agree that subclinical 25-D deficiency is common, particularly 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 sufficient. Serum concentrations of 25-D are known to vary with age, sex, race, season and geographic location. 1 This has led many to establish seasonal expected ranges for their geographic location and local population. This approach provides 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 nutritional threshold, below which an individual could be characterized as vitamin D deficient. Several investigators 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 hormone levels. 10 Secondary 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 subsequent skeletal density. 10 Further studies have shown that 25-D levels of less than 32 ng/ml are associated with impaired insulin resistance and beta-cell function. 10 Together, these data suggest that 32 ng/ml represents the appropriate threshold for identifying individuals with clinical vitamin D deficiency. Laboratory Measurement of Vitamin D Laboratory assays are available for the measurement of both 25-D and 1,25- D. Assays for 25-D are generally 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 hormone, 1,25-D. Since circulating levels of 25-D closely reflect the total amount of web.com advance /LABORATORY FEBRUARY

6 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. 2,6 In fact, levels of 1,25-D can often be normal in individuals with overall vitamin D deficiency. 6 Measurement 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 increasingly 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 manufactured by irradiation of ergosterol from yeast to produce ergocalciferol, or vitamin D2. Both the D2 and D3 forms have similar biological activity. 2 In the United States, vitamin D deficiency is commonly treated with vitamin D2 in the form of Drisdol, while both D2 and D3 are used to fortify foods. 11 These two parent compounds 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 vitamin 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 clinician to the erroneous conclusion that additional supplementation is required, resulting in vitamin D With the growing number of diseases associated with its deficiency, the accurate measurement of vitamin D levels has become increasingly important. intoxication and hypercalcemia. 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 available methods in the United States fail to measure both components in an equimolar manner. 11 The measurement of 25-D has evolved from the cumbersome and labor-intensive procedures developed in the 1970s to today s highly automated methods performed on random-access analyzers. 11 Several early assays used the naturally occurring vitamin D-binding protein (DBP) and required extraction or chromatographic purification as pretreatment steps. 11 Unfortunately, in the absence of extensive extraction and/or chromatographic sample preparation, DBPbased assays were plagued with significant interference and cross-reactivity problems. 11 These assays were generally replaced by the more reliable immunoassays that employ highly specific monoclonal antibodies to vitamin D. 11 Automated chemiluminescence-based assays have become commercially available, using either the DBP11,12 or a specific antibody. 11,13 Recent studies indicate, however, that the DBP-based automated 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 1. Holick MF, Jenkins M. The UV Advantage: The Medical Breakthrough that Shows How to Harness the Power of the Sun for Your Health. New York, NY: ibooks; Endres DB, Rude RK. Mineral and bone metabolism. In: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia, PA: W.B. Saunders; 1999: National Institutes of Health, Office of Dietary Supplements. Dietary Supplement Fact Sheet: Vitamin D. Available at: gov. Accessed 07/14/ Holick MF. Vitamin D: A millennium perspective. J Cell Biochem Feb 1; 88(2): [Mellanby E.] Nutrition Classics. The Lancet 1:407-12, An experimental investigation of rickets. Edward Mellanby. Nutr Rev. 1976; 34(11): Reprint. 6. Zittermann A. Vitamin D in preventive medicine: Are we ignoring the evidence? Br J Nutr. 2003;89(5): Holick MF. Vitamin D: Importance in the prevention of cancers, type 1 diabetes, heart disease, and osteoporosis. Am J Clin Nutr. 2004;79(3): Krause R, Buhring M, Hopfenmuller W, Holick MF, Sharma AM. Ultraviolet B and blood pressure. Lancet. 1998;352 (9129): 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): Hollis BW. Circulating 25- OH D levels indicative of vitamin 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): Roth HJ, Zahn I, Alkier R, Schmidt H. Validation of the first automated chemiluminescence protein-binding assay for the detection of 25-hydroxycalciferol. Clin Lab. 2001;47: Ersfeld DL, Rao DS, Body J-J, et al. Analytical and clinical validation of the 25 OH vitamin D assay for the LIAISON automated analyzer. Clin Biochem. 2004;37(10): Binkley N, Krueger D, Cowgill CS, et al. Assay variation confounds the diagnosis of hypovitaminosis D: A call for standardization. J Clin Endocrinol Metab. 2004;89(7): advance /LABORATORY FEBRUARY web.com

7 2005 Laboratory Corporation of America Holdings All Rights Reserved L