Welcome to today's session on vitamin D in the era of precision medicine. My name is Felice Caldarella. Now we'll be serving as your moderator today. We'll be hearing a lecture from Dr. Michael Levine on this very fundamental subject. Dr. Levine is Chief Emeritus of Endocrinology and Diabetes and Director of the Center for Bone Health at the Children's Hospital of Philadelphia. Dr. Levine holds the Lester Baker Endowed Chair and is Professor Emeritus of Pediatrics and Medicine at the University of Pennsylvania Permanent School of Medicine. Dr. Levine's research interests focus on the genetic basis of the endocrine diseases that affect bone and middle metabolism, particularly rickets, primary hyperparathyroidism and hypoparathyroidism. Dr. Levine has published over 400 manuscripts, chapters and reviews. He's a former editor of the Journal of Clinical Endocrinology and Metabolism and has served as a member of the Board of Directors of the US Pediatric Endocrine Society. He's received numerous awards in recognition of his accomplishments as a physician scientist, including the Distinguished Endocrinology Award for the American College of Endocrinology, the Frederick C. Barter Award from the American Society of Bone and Middle Research and the International Award from the European Society for Pediatric Endocrinology. Dr. Levine. Greetings from Philadelphia. I want to thank the organizers for inviting me to talk with you today. My goal will be to introduce the notion that vitamin D therapy can be improved by the principles of precision medicine. I have nothing to disclose that would represent a conflict of interest and I'd like to review with you my learning objectives for this talk. At the conclusion of my talk, you should be able to describe the effects of vitamin D. You should be able to assess the environmental and genetic bases for vitamin D deficiency and you should be able to formulate a personalized treatment plan for vitamin D deficiency. So let me first introduce the topic of precision medicine, which has been the subject of a great deal of interest over the past decade. And as many of you know, it's an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment and lifestyle for each person. And precision medicine is a step towards personalized medicine in that it uses more variables to stratify patients but is not completely individualized. And lastly, significant advances have been made but precision medicine is not yet the standard practice for many diseases. This slide shows the evolution of practice towards precision medicine. Today, we are beginning to move beyond the one size fits all philosophy in which disease treatment and prevention strategies are developed for the average person with less consideration for the differences between individuals. In many cases, we have already incorporated a stratified medicine approach that cohorts patients into treatment groups based on demographics, clinical features and even some biomarkers. The ultimate goal of course is precision medicine, which also takes into account more individual variability such as variability in medications, behaviors, environment and lifestyle, as well as variability in genes that may influence an outcome for each person. This approach will allow doctors and researchers to predict more accurately which treatment and prevention strategies for a particular disease will work best in which groups of patients. Now, to answer the question today of whether precision medicine can guide our approach to vitamin D therapy, I will address the following three questions. What are the effects of vitamin D deficiency? What are the conventional factors that influence vitamin D homeostasis? And do genes influence vitamin D metabolism? The Institute of Medicine and several other medical societies have proposed that a serum 25 hydroxyvitamin D level of 20 nanograms per ml is sufficient for nearly 98% of the healthy population. And that a level of less than 12 nanograms per ml represents vitamin D deficiency. And despite this pronouncement, there is still much controversy. Professional societies such as the Endocrine Society and the American Society for Bone and Mineral Research have pushed back a bit against these recommendations and offered counter proposals that 30 nanograms per ml represents sufficiency. And this higher target is based on the presumed needs of patients with metabolic bone disease and other patients who have nontraditional disorders that have been associated with low concentrations of 25 hydroxyvitamin D in observational studies. Overall, however, I think we can agree that there's a growing consensus to support a range of 20 to 50 nanograms per ml as most desirable for people and not think about vitamin D sufficiency with a critical cutoff. So how common is vitamin D deficiency? Using the Institute of Medicine targets, these most recent data from NHANES show that the prevalence of vitamin D deficiency defined as a 25 hydroxyvitamin D of less than 12 nanograms per ml and shown by the blue bars is common in the adult population, especially among young adults in whom there can be nearly a 20% risk of vitamin D deficiency. In addition, we can see in the orange bars evidence of vitamin D insufficiency, and this can be as high as nearly 40% in non-Hispanic blacks. And vitamin D deficiency can be as high as 18% in non-Hispanic blacks. So it's clear that the risk of vitamin D deficiency does not extend in equal fashion across the entire U.S. population and that people of color are at greater risk for vitamin D deficiency than patients who are non-Hispanic white. Leaving aside for the moment studies showing statistical associations between low blood concentrations of 25 hydroxyvitamin D and various conditions, it's clear from much evidence that the primary clinical manifestations of vitamin D deficiency are rickets and osteomalacia, which reflect the critical impact of vitamin D and vitamin D deficiency on bone and mineral metabolism. Rickets affects function of a growth plate, while osteomalacia affects mineralization of the entire skeleton. The skeleton is the principal target organ of vitamin D and vitamin D deficiency with bone pain, pseudo fractures and skeletal deformities such as bowed or knocked knees, very common in growing children and bone deformity also common in older adults with vitamin D deficiency. Because vitamin D affects the growth plate, we see decreased linear growth in children with vitamin D deficiency. Other important features of vitamin D deficiency include hypotonia and muscle weakness due to hypocalcemia and hypophosphatemia. We also see developmental delays with delayed walking and delayed eruption of teeth in growing children. And there's increased respiratory infections in children and adults, perhaps due both to impaired innate immunity from vitamin D deficiency and also weakened chest muscles that hamper the ability to clear bronchial secretions. Now, the consequences of vitamin D deficiency reflect disturbances in the classical physiologic actions of vitamin D. And in the absence of vitamin D action, there is reduced gastrointestinal absorption of calcium and phosphorus. There's also decreased mobilization of calcium and to a lesser extent phosphorus from skeletal stores. And with the development of hypocalcemia in concert with decreased vitamin D action, there's increased synthesis and secretion of parathyroid hormone. And then increased levels of PTH affect the kidney where they decreased reabsorption of phosphate. Now, these defects in vitamin D action result in important and predictable biochemical profile, which intensifies through three stages of worsening vitamin D deficiency. And they're really characterized by increasing evidence of vitamin D deficiency by levels of 25-hydroxyvitamin D that become lower and lower and really quite low by stage three. The critical point is the development of secondary hyperparathyroidism. And that can occur very early and will normalize serum calcium levels so that in stage two, we may see normal levels of serum calcium, even though levels of PTH are elevated. Levels of bone-specific alkaline phosphatase may be the only signal in older adults who have vitamin D deficiency. What's critically important here is that the development of secondary hyperparathyroidism leads to decreased renal tubular reabsorption of phosphorus, which leads to worsening hypophosphatemia. And I stress this because most of the CMP, Comprehensive Metabolic Panels, or SMAC profiles that we order do not include a serum phosphorus. So it's important to think about serum phosphorus when evaluating a patient for metabolic bone disease, and in particular, vitamin D deficiency, and to make sure and order a serum phosphorus, as well as the SMAC or CMP profile. Low serum phosphorus levels lead to a disorganization of the growth plate in children and defective mineralization of cartilage and osteoid in children, as well as adults that causes osteomalacia. In children, radiologic evidence of rickets, again, a disease of the growth plate, is most obvious at the ends of the most rapidly growing long bones, such as the knee and the wrist, as shown here. And we can see widening of the space between the metathesis and the epiphysis. So this is the physis or growth plate, and there's widening of this gap when there is rickets, and that's an important finding. There's also fuzziness of the growth plate due to decreased mineralization and splaying, and here you can see cupping because of the softness of the metathesis. And all of this leads to weakness of the bone and greater tendency to deformity of bone. And then decreased mineralization throughout the skeleton leads to a general softening of the bones that twist, and it's thought that this has given rise to the term rickets, perhaps from the Old English word ricken, which means to twist, although I must admit that this etymology is only conjecture. Osteomalacia in adults is far less dramatic than rickets in children. Adults may have generalized musculoskeletal pain, a waddling gait, and lower extremity deformities. There is generalized demineralization of the skeleton with trabecular bone loss that mimics osteoporosis, and remember, a DEXA scan cannot distinguish between osteomalacia and osteoporosis. In osteomalacia, there can be fractures and pseudo-fractures or looser lines, as shown here by the white arrows in both insets that represent stress fractures and often appear symmetrically in the inner femora, as well as in the inferior pubic rame, as well as in ribs, shown here by the black arrow. Pseudo-fractures often progress to complete fracture with minimal trauma. And what about possible non-bone diseases and vitamin D, the so-called nontraditional effects of vitamin D? Many observational studies have suggested disease associations with low serum levels of 25-hydroxyvitamin D, such as type 1 diabetes or type 2 diabetes, heart disease, cancer, infections, including COVID-19. Nevertheless, randomized clinical trials have produced inconsistent evidence of benefit of vitamin D supplementation in the patients with these conditions. And so the evidence is inconclusive about the effect of treatment with vitamin D on physical functioning and on many other disorders that have been associated with low levels of 25-hydroxyvitamin D. So coming back to our questions, having addressed what the effects of vitamin D deficiency are and what is vitamin D deficiency, let's take a look at the risk factors for vitamin D deficiency. Having defined rickets as the principal manifestation of vitamin D deficiency, we can trace the epidemiology of rickets to identify the risk factors for vitamin D deficiency. And urban migration in the 18th century is a great starting point because this urban migration led to a remarkable increase in the prevalence of rickets, particularly among the poor who lived in crowded city tenements. This was associated with a lack of direct sunlight exposure at street level due to narrow streets, as you can see here, and tall buildings and large, dark shadows. Sunlight was also blocked by heavy smoke that choked the skies. So why is a lack of sunlight associated with rickets or vitamin D deficiency? The link between sunlight and vitamin D homeostasis was discovered in the early 20th century. And over the next decades, the whole story was illuminated, and you'll pardon my pun. Let's review vitamin D metabolism. Generation of vitamin D in the skin, in the upper layers of the epidermis, requires UVB irradiation, 280 to 315 nanometer. Solar radiation is most effective when the sun is at an angle higher than 50 degrees above the horizon, which occurs from 10 a.m. to 3 p.m. each day. And direct, not reflected sunlight that is typical at street level in cities with tall buildings is needed. Solar irradiation is also less effective during winter months at greater distances from the equator due to the more acute angle of the winter sun. UVB radiation leads to photo isomerization of 7-D hydrocholesterol to pre-vitamin D3. And at body temperature, pre-vitamin D is converted to cholecalciferol, vitamin D3. Vitamin D intoxication is prevented by isomerization of excess pre-vitamin D to inactive photo products. Now, vitamin D made in the skin or obtained from the diet in the form of cholecalciferol or vitamin D2 ergocalciferol must first undergo sequential hydroxylations to become fully active as the hormone calcitriol, 1,25-dihydroxyvitamin D. And there are two important hydroxylations. First, in the liver, CYP2R1, the principal 25-hydroxylase, converts parent vitamin D to 25-hydroxyvitamin D. And then in the kidney, under the required action of PTH, 25-hydroxyvitamin D undergoes a hydroxylation at the one position to become 1,25-dihydroxyvitamin D. And this is the fully active form of vitamin D and is considered a hormone because it binds to the vitamin D receptor. Now, in addition to skin pigmentation, vitamin D deficiency can also occur when clothing or sunblockers prevent UVB irradiation from reaching the skin. And of course, thin skin, as occurs in aging individuals, is less efficient. Other risk factors include poor intake or absorption of dietary vitamin D. And blocks in activation of vitamin D can also reduce vitamin D action. For example, liver or renal disease can impair hydroxylation and full activation of vitamin D. And lastly, another barrier to vitamin D deficiency can be increased loss of vitamin D, for example, which occurs via renal excretion of the vitamin D binding protein in patients with nephrotic syndrome. In addition to the conventional limitations to healthy vitamin D homeostasis, we have also become aware that there are additional challenges. Vitamin D deficiency is also common among the very overweight and the elderly. In addition to fat sequestration of vitamin D in abundant adipose tissue, Jeff Roizen, when a fellow in my lab and now an assistant professor at Penn, showed that expression of CYP2R1, which encodes the principal hepatic 25-hydroxylase, is reduced in obese mice. He found similar effects in aging mice. We believe that reduced expression of CYP2R1, the 25-hydroxylase, may also contribute to lower circulating levels of 25-hydroxyvitamin D in obese and aged humans as well. And it's important to recognize that certain medications, such as first-generation anticonvulsants, have been associated with impaired vitamin D homeostasis via induction of hepatic P450 enzymes, such as CYP3A4. What about genetics? Vitamin D status and the response to oral vitamin D supplements are highly variable, even among normal subjects. The figure on the right shows tremendous variability in the basal concentration of serum vitamin D, 25-hydroxyvitamin D, in 91 post-menopausal women. And these baseline concentrations are shown in the solid circles. And these post-menopausal women showed highly variable responses to supplementation with oral daily vitamin D3, 2,300 units or 2,500 units, for four to six months. And this is remarkable variability in their responses in healthy individuals. We would ask, can genetic variability account for this remarkable variation, not only in basal levels, but also in the response to vitamin D supplementation? So this leads me to the third question for today. Do genes influence vitamin D metabolism? Let's take a look at the evidence that genetic variability can influence vitamin D status. Workers have used genome-wide association studies, GWAS, to identify genes in which variant alleles are associated with serum levels of 25-hydroxyvitamin D in the normal population. Here we see, for example, four important genes outlined in green. First, DHCR7, which causes Smith-Lemley-Opitz syndrome, is certainly associated with serum levels of 25-hydroxyvitamin D. In patients who have Smith-Lemley-Opitz, the loss of DHCR7 activity results in production of less cholesterol with increased availability of 7-dehydrocholesterol to become cholecalciferol. And these patients actually have higher levels of 25-hydroxyvitamin D. Another important gene is the GC protein gene, and this encodes the principal vitamin D binding protein that transports vitamin D metabolites in the circulation. And it makes sense that levels of GC protein would be strongly associated with serum levels of total 25-hydroxyvitamin D, just as levels of levothyroxine or T3 are strongly associated with differences in circulating levels of thyroid-binding globulin. And then we have CYP24A1, which encodes the principal enzyme that inactivates vitamin D metabolites, including 25-hydroxyvitamin D. Patients who have loss-of-function mutations in 24A1 have elevated serum levels of 25-D and 125-dehydroxyvitamin D. And these patients develop hypercalcemia and or hypercalceria, a condition termed idiopathic infantile hypercalcemia. But of all these genes, our interest has been greatest in CYP2R1, which we had previously identified as the principal 25-hydroxylase enzyme that converts vitamin D to 25-hydroxyvitamin D. And as I've already told you, expression of CYP2R1 is reduced in aging and obesity, which accounts in part for lower serum levels of 25-hydroxyvitamin D. In collaboration with Tom Thatcher, then working in Nigeria, we found that children who carry CYP2R1 mutations have low serum levels of 25-hydroxyvitamin D. And here you can see patients who are homozygous for these mutations and just how low their baseline levels are. And we went ahead and tested the responsiveness to vitamin D supplements in children who were homozygous or heterozygous, shown here in the light blue line, for CYP2R1. In red are shown the 95% confidence limits of serum 25-hydroxyvitamin D in normal children who were given an oral bolus dose of 50,000 units of vitamin D. Children with the biallelic CYP2R1 mutations shown in the yellow line showed almost no response to administration of vitamin D in terms of their 25-hydroxyvitamin D levels. But perhaps even more remarkable are the responses of children who are heterozygous for CYP2R1 mutations, who are shown in the light blue line. These patients also have somewhat lower baseline serum levels of 25-hydroxyvitamin D and show a response, but a response to vitamin D supplementation that is not quite normal. These responses show a clear gene dose effect on production of 25-hydroxyvitamin D and raise the possibility that some patients with lower levels of serum 25-hydroxyvitamin D and poor responses to vitamin D supplementation might be heterozygous for a non-functional CYP2R1 allele. We wanted to ask the question, what about CYP2R1 variants in the normal population, the patients that we see all the time who might have slightly low levels of 25-hydroxyvitamin D? Here, Jeff Roizen asked whether CYP2R1 genotype in a normal population might indicate a contribution of CYP2R1 to the variable responses to vitamin D supplementation that are seen in children and adults. The panel on the left shows a wide range of changes in serum 25-hydroxyvitamin D levels after supplementation of normal children with vitamin D3, either given no supplementation or daily doses ranging from 400 to 4,000 units a day for 12 weeks. Jeff contacted Dr. Lewis, who had done this study, and obtained DNA samples from the patients. And he went ahead and genotyped a CYP2R1 polymorphism that he had identified in the promoter of the gene and which he had found that was associated with decreased expression of CYP2R1 in vitro. Jeff studied 160 patients whose increases in 25-hydroxyvitamin D after supplementation were in the lowest and the highest quartiles. The data are shown on the right. No one in this sample was homozygous for the T allele, which is the allele that corresponds to decreased expression of CYP2R1. The T allele was present in nearly 40% of African-Americans but in only 19% of Caucasians in this sample. And of greatest interest, if you look at those individuals who had the most robust responses to vitamin D, whose 25-hydroxyvitamin D levels were in the highest quartiles, none of those patients carried the T allele at all. So these results suggest that at least part of the variability and responsiveness to vitamin D supplementation in a normal population may be genetically determined by a polymorphism within CYP2R1. Well, there are many publicly available genomic databases, and Alex Casella, working in our lab and now an MD-PhD student at the University of Maryland, analyzed a number of these publicly available databases and saw that the CYP2R1 gene is a highly polymorphic gene in the general population. Many normal individuals carry allelic variants that distinguish them from the reference allele. She went ahead and characterized the functional capacity of each of these allelic variants that coded for CYP2R1 proteins that had amino acid substitution. And again, I'll remind you, these are present in the normal population. And after expressing each of these variants in HEK293 cells and analyzing the 25-hydroxylase activity of the specific recombinant proteins, she found that many of these allelic variants encoded for CYP2R1 proteins that had reduced function shown in the red circles. In addition, what was, I think, even more surprising was that several of these variants actually had increased CYP2R1 activity. And so looking at normal individuals, we can see that there's tremendous variability from individual to individual in the CYP2R1 genotype. And based on functional differences in these allelic variants, we presume that there is a contribution to the baseline and response of 25-hydroxyvitamin D to D supplementation based on a particular variant, a normal individual carries. Now, Alex also looked at the prevalence of a large number of these variant alleles, particularly those that had reduced enzyme activity in different normal populations around the world, as shown on this map. And Alex found that the alleles that had reduced activity or even no activity were far more common in high-sun areas around the equator than in low-sun areas, similar to the distribution of darker skin. She also found that latitude significantly predicts the population variability in null allele frequency. Now, these results suggest that genetically decreased CYP2R1 activity might be an adaptive response to high-sun exposure near the equator and might represent one of the mechanisms that we use to avoid vitamin D intoxication. Now, what about genetic changes that might affect inactivation of vitamin D? The CYP2R1 gene is the principal enzyme that inactivates 25-hydroxyvitamin D and clearly is strongly associated with serum levels of 25-hydroxyvitamin D. But there is a second enzyme present in the liver and in the intestine, CYP3A4, that can also inactivate vitamin D metabolites. Now, why be interested in CYP3A4? Well, it's the most abundant P450 enzyme in the liver and metabolizes most drugs, xenobiotics, and many steroid hormones, including vitamin D. And there's high variability in CYP3A4 expression, and this variability is caused by non-genetic as well as genetic factors and contributes to unpredictable responses to drugs and unpredictable drug toxicities. And importantly, induction of CYP3A4 by drugs such as phenytoin or antiretroviral drugs can lead to vitamin D deficiency, something we've noticed for many, many years, but perhaps hadn't joined the dots and recognized that the action of these drugs was mediated through induction of CYP3A4. And lastly, a few years ago, Jeff, working in our laboratory, identified a recurrent gain-of-function mutation in CYP3A4 that was associated with rickets and poor responsiveness to vitamin D therapy in patients who carried this mutation. And the mutant CYP3A4 that Jeff identified inactivates vitamin D metabolites in vitro, and we were able to show that it also could inactivate vitamin D metabolites more rapidly in vivo. The blue line in this graph shows the robust response of normal subjects to an oral dose of 50,000 units of vitamin D3. By contrast, subjects with the CYP3A4 mutation shown in red have a remarkably blunted response consistent with rapid inactivation of 25-hydroxyvitamin D. And importantly, similar effects occur in patients taking phenytoin, phenobarbital, antiretroviral drugs, or even rifampin, all drugs that induce expression of CYP3A4. From what I've presented so far, it should be obvious that patients who have medical conditions or take medications that can affect vitamin D metabolism may not be the kinds of patients for whom a one-size-fits-all approach is appropriate. In addition, many patients will carry gene variants that can also affect vitamin D homeostasis. So we must ask ourselves, based on these patients having conditions, taking medications and genetic variants that affect vitamin D needs, can we use precision medicine in order to be more precise about screening and treating vitamin D deficiency? A lack of benefit of vitamin D supplementation for non-bone diseases has led the U.S. Preventative Services Task Force to recommend against routine measurement of 25-hydroxyvitamin D as a screen for vitamin D deficiency in otherwise healthy individuals. But I think it's important to also realize that the current recommendations for screening for vitamin D deficiency do not apply to individuals who are hospitalized or institutionalized or to those patients who have underlying conditions, for example, osteoporosis, osteomalacia, malabsorption, cystic fibrosis, morbid obesity, or who take medications, all of which would increase the risk of vitamin D deficiency. Now, looking at a one-size-fits-all approach, most individuals will do fine following the IOM guidelines for daily vitamin D intake, but many patients will not be able to show vitamin D sufficiency following these requirements. I want to emphasize that all babies and infants under 12 months of age need 400 units of vitamin D daily, whether they're fed formula or breast milk. And from age one to 70, the RDA for the general population has been set at 600 units a day. But having said this, I must repeat myself, these recommendations do not apply to everyone. So precision therapy with higher daily doses of vitamin D and regular monitoring of serum 25-hydroxyvitamin D concentrations will benefit many of our patients. The breastfed infant who does not reject vitamin D supplementation is a particularly vulnerable patient. But in this case, nursing mothers who are not giving their baby 400 units a day of vitamin D can fortify their milk to contain about 400 units of vitamin D per quart. I call this super milk. And they can do this by taking a daily vitamin D supplementation. And 400 units a day shown in these red boxes will not do it. 2,000 units a day shown in the open circles will not do it. But 5,000 to 6,000 units a day taken by the mother will fortify her breast milk and her breast milk will be comparable to infant formula. And this will be safe for the mother and therapeutic for the infant. What are some other examples of how precision medicine can guide us with vitamin D? How about patients with decreased expression or activity of hepatic CYP2R1? Not just patients who have uncommon genetic mutations, but patients with severe fat malabsorption or liver disease or biliary atresia or patients with morbid obesity and aging, both conditions that reduce expression of CYP2R1. Well, these patients may benefit from administration of larger doses of the calciferols, cholecalciferol, or they might respond rather well to standard doses of calcifediol, 25-hydroxyvitamin D. And calcifediol is more water-soluble than the calciferol parent compounds, cholecalciferol or ergocalciferol. So the calcifediol is better absorbed, and because it already is 25-hydroxylated, it bypasses the defect in the liver. And what about patients who have gain-of-function mutations in CYP3A4 or high-expressing genetic variants or patients who are taking medications that induce CYP3A4, such as anticonvulsants like dilantin or phenobarbital or rifampin or even St. John's wort? These patients will have increased inactivation of vitamin D metabolites. In this case, one could give higher doses of calcifediol or even calcitriol, and I think these patients should have serum 25-hydroxyvitamin D levels monitored closely. And lastly, patients with renal disease or mutations in CYP27B1, which encodes the 1-alpha-hydroxylase or patients who have conditions such as hypoparathyroidism in which an absence of PTH prevents conversion of 25-hydroxyvitamin D to 125-dihydroxyvitamin D. Well, here it makes perfect sense to provide the fully active form of vitamin D calcitriol. And so in conclusion, let me say that vitamin D deficiency remains a very common challenge within the normal population as well as within patients who have underlying conditions that impair absorption of vitamin D or activation of vitamin D. I'll remind you that despite all the hoopla of association studies that have shown some relationship between levels of 25-hydroxyvitamin D and a whole host of nontraditional conditions, the primary manifestation of vitamin D deficiency is metabolic bone disease. I want to remind you again that you should consider disease-associated risk factors that affect vitamin D supply and activation when choosing a vitamin D therapy scheme, introducing precision medicine into your approach to screening and treating vitamin D deficiency. And lastly, there's emerging knowledge regarding genetic variants that affect vitamin D metabolism. And as we learn more about these variants, that knowledge will enhance our ability to use precision medicine to guide vitamin D therapy. I'd like to thank you for your attention. I'd also like to acknowledge my many collaborators at CHOP and the University of Pennsylvania who've assisted me in performing these studies, many of which I've shared with you today, also my collaborators at the Mayo Clinic and the University of Washington. And I'll leave you with one final question, to D or not to D? That is the question. Thank you very much for your attention. Welcome. Now it's time for our question and answer. And to D or not to D, that is the question before us. And thank you, Dr. Levine, for your presentation on vitamin D in the era of precision medicine. We have questions. And so I think we'll just start right in. The first question at the top is, what is the minimal time after which we may have a response after oral or injectable therapy? We're seeing a lot of questions about proper vitamin D regimen. So Dr. Levine, can you take it? It's a pleasure to be here with you this morning. I've been monitoring the questions as they've come in, and there's a great many important and interesting questions. First, let me address this one. We don't have any parenteral forms of cholecalciferol, which is D3, or ergocalciferol D2, in the United States. So we have to look at old studies in order to get some idea of the timing onset. If you give IM vitamin D, 500,000 or 600,000, you'll certainly see an increase in serum phosphorus, which is the first sign of reversing vitamin D deficiency in about a week. If you gave 500,000 or 600,000 units of vitamin D orally, you would see about the same time response. This is much shorter than the response you would see giving 5,000 units a day to a patient with vitamin D deficiency. What I typically do is give a loading dose of 50,000 to 100,000 units of Coley-Calciferol. This is easy to do because you can get 50,000 unit capsules, so one or two taken immediately. That's the acute treatment. And then I follow that up with 50,000 units once weekly for 10 to 12 weeks. You need to treat for about 10 to 12 weeks because the half-life of 25-hydroxyvitamin D is about two weeks, and so we need to use about 10 to 12 weeks to get to a true steady state. Patients who have severe vitamin D deficiency, so a 25-hydroxyvitamin D level of less than 12, will require in general about a half million units for full replacement. So you could achieve that by giving Stas therapy, 500,000 units once or 250,000 units on two different days, or you could achieve it by 50,000 units a week for 10 weeks or 5,000 units a day. So you've got tremendous flexibility based on what you think the patient's compliance is going to be. But the large bolus dose certainly gets things started much more rapidly, which is why I do a hybrid model of a loading dose and then weekly dose. And once you've done that and you've done the 10 to 12 weeks, you're rechecking vitamin D levels. Any other, and what's your goal of vitamin D level at that point, and are you checking any other labs, magnesium, PTH? Great, great questions. I think PTH is very helpful. It's sort of like looking at TSH to guide you as you replace levothyroxine, thyroid hormone supplements, and I actually follow patients monthly to make sure that they're responding the way I expect them to respond. I also do an evaluation before I treat to make sure I'm not missing celiac disease or other underlying disorders that might account for malabsorption. So on a monthly basis, I will check a serum calcium, phosphorus, and alkaline phosphatase. That's the so-called holy trinity of metabolic bone disease, serum calcium, phosphorus, and alkaline phosphatase. I'll check the 25 hydroxy vitamin D level. And so that will be what I check every month, and then at the end of the 10 to 12 weeks, I'll want to make sure that all of that is normal as well as the PTH. I don't look at 125 levels because it's not very helpful. It's difficult to interpret, and I'll try to achieve a 25 hydroxy vitamin D level that is greater than 20 in somebody who doesn't have an underlying metabolic bone disease. And if they do have an underlying metabolic bone disease such as osteoporosis, I'll shoot for a level that's greater than 30. So somewhere between 20 and 50 is what I think is the sweet spot for serum 25 hydroxy vitamin D. 20 is fine for otherwise normal individuals. For somebody with osteoporosis, most authorities would recommend 30 as your lower limit for a target. And how about any calcium supplementation on your patients while you're treating their vitamin D? So we certainly want to make sure that a patient is receiving the recommended daily allowance of calcium and encourage people to achieve this through their diet. And only if their diet cannot provide them with the RDA, which in most adults is going to be 1,000 to 1,500 milligrams a day of elemental calcium. If they can't achieve this through their diet, then we would recommend supplements. If somebody's serum calcium level is low when you begin therapy, I would probably give an additional 1,000 milligrams of elemental calcium per day. But if their serum calcium level is normal, I think the RDA would be satisfactory. Let me also say that if the bone alkaline phosphatase was elevated, even in the presence of a normal serum calcium, I would also give additional calcium supplementation. And this is because if the alkaline phosphatase is elevated, it tells you there's a mineralization defect in the bone. If the serum calcium level is low, it tells you there's a deficit of calcium in the body. And in either of those two conditions, as you begin to replace with supplemental vitamin D, you can cause a rapid remineralization of bones, the so-called hungry bones effect. And this can worsen pre-existing hypocalcemia or even cause hypocalcemia in a patient who has a normal serum calcium level. So additional calcium beyond the RDA if the serum calcium level is low or if the bone alkaline phosphatase is elevated. Great, thanks. In your presentation, you talked about medications that can contribute to low vitamin D levels, like the first generation, antielectics, phenobarb, and dilantin. There's a question here about reassessing the need of loop diuretics, which are sometimes being used for edema, and to consider alternatives. And that this particular provider sees a lot of secondary hyperpara, which corrects with the discontinuation of calcium supplementation. Can you talk more to that question? Well, I'll tell you, it's a great point. Where I see the effect of loop diuretics on mineral metabolism is in our NICU, where nearly all the babies are on loop diuretics. They almost all have mild hypercalciuria because of this. Many of them will go on to develop nephrocalcinosis, and many of them are losing so much calcium through their urine that they develop secondary hyperparathyroidism. So if a patient needs a loop diuretic, they have severe underlying cardiac disease. Sometimes you can add a thiazide to the loop diuretic to mitigate some of the urinary calcium loss. In other cases, that might not be possible, and then you'll have to give additional calcium and vitamin D in order to reduce the PTH level and mitigate the secondary hyperparathyroidism. And endocrinology is tough, it's complicated. If you can't reduce the urinary calcium excretion, then you have to address the secondary hyperparathyroidism, but realize that that's going to worsen the hypercalciuria, but you have to pick your battles. These are tough situations. There's a question here about using the free 25-hydroxyvitamin D level for any difference between taking daily versus weekly vitamin D. Let me address the free 25-hydroxyvitamin D question first. In most of endocrinology, we cling to the hypothesis of the free hormone hypothesis, which says that the biologically active form of the hormone is the free form. With vitamin D, things may not be so clear. For example, in order for vitamin D to get into the kidney and for 25-hydroxyvitamin D to get into the kidney and become 125, it has to be attached to the vitamin D binding protein. So free 25-hydroxyvitamin D doesn't diffuse across the membrane of the proximal renal tubular cell well. It's mostly brought in by endocytosis of the vitamin D binding protein. On the other hand, in monocytes, where vitamin D has an immunoregulatory role, it is the free vitamin D, the free 25-hydroxy that diffuses across the membrane. And it may be the free 25-hydroxyvitamin D that diffuses across the membrane of the parathyroid cell and contributes to regulation of PTH secretion in that cell. So it's not perfectly clear what is the most important component, whether it's free 25-hydroxyvitamin D or the total. But let me say this. There's one patient that was described several years ago in the New England Journal of Medicine who had a genetic defect in vitamin D binding protein. She had no circulating vitamin D binding protein. Her levels of total 25-hydroxyvitamin D were very, very low. Her levels of free 25-hydroxyvitamin D were low normal. And she didn't have secondary hyperparathyroidism or any obvious consequence of having no vitamin D binding protein. So maybe we really have to relook at the free hormone hypothesis, even in the field of vitamin D. Having said that, from a clinical point of view, there doesn't seem to be any significant advantage to measuring free 25-hydroxyvitamin D compared to total 25-hydroxyvitamin D, with the possible exception of a patient with severe liver disease and very low levels of vitamin D binding protein, where knowing the free level is normal may provide some degree of reassurance. I would say that we could probably look to the PTH to tell us whether or not 25-hydroxyvitamin D was adequate in that patient, because just as we look at TSH to tell us something about thyroid hormone status in a patient with low levels of thyroxine binding protein, if the PTH is normal in a patient with severe liver disease and they have a low 25-hydroxyvitamin D, the free level is probably normal. And if you could measure it, that would be the one situation where it would be helpful. Yeah, great answer. I like especially using the PTH. It's sort of like the internal barometer for vitamin D in those cases. So we have actually less than a minute to go. There is an interesting question here from Dr. Trentz regarding the association of elevated vitamin D levels and cardiovascular disease. So interesting. Yeah, so I think that the large registries databases from Scandinavia have looked at overall mortality and cardiovascular mortality as a function of serum concentration of 25-hydroxy vitamin D. And these studies are epidemiological. They're not clinical trials giving people vitamin D and looking at mortality in the following years. They're cross-sectional, but they show a U-shaped relationship so that mortality is higher when levels of 25-hydroxyvitamin D are less than 12, and mortality is slightly higher when 25-hydroxyvitamin D levels are greater than 50. So I think for everyone, you want people in that sweet spot between 20 and 50, because at least from epidemiological studies, that seems to be the optimal range for 25-hydroxyvitamin D levels. And no better answer to our talk today on precision medicine to help us guide us in vitamin D replacement. So it looks like our time this morning has ended. I would like to thank Dr. Levine for an excellent presentation and answering your questions, and to the audience for their questions. Sorry we couldn't get to them all, but definitely we'll hear more about vitamin D. The word on vitamin D hasn't ended. So again, thank you, Dr. Levine. Thank you to the presenters, and everyone have a good afternoon. Everyone take care and be safe. Greetings, everyone. What a great meeting this was, and now comes the hard part for me at least. As I approach the end of my presidential term at ACE, I cannot help but look back and think about what a year this has been. A year unlike anything ACE has seen before. ACE has undergone a complete transformation over the last several years, and this last one has been the most dramatic, and not just because of a pandemic. This past year, we have really seen us reflect on what ACE is, and how we can be even better. We asked our members what they want, and they have responded. We have transformed our brand, including how we look. We have upgraded our infrastructure to better serve all of our community, and have reached out to collaborate with new partners. We have expanded our membership to include those we work with and lead, and those we treat. We have seen a sea change in where our finances are coming from, and ACE remains strong and competitive. And as I said in the beginning, we have worked hard to shift our focus to strategic priorities that our community has told us they care about, including knowledge and education, publications and guidelines, community engagement, and patient and public awareness. In these areas, we have already started developing critical programs and efforts that are sure to greatly benefit both members and patients in the years ahead. To do this takes all of us, especially a great team of staff, ELT, officers, board members, committee members and chairs, DSN members, and especially volunteers. Without all of you working harmoniously, none of these achievements would have been possible. The time spent and energy has been incalculable, but the results superb. No leadership could have asked for more, especially in the face of all the hardships all of us have endured. What a relief it will be when Zoom actually means how fast we are going. This does not mean that the year has been without its disagreements and disputes. Not everyone will agree with change and how it progresses, but change is inevitable. In fact, without it, all of us will stagnate and die, including associations. One has to constantly look at ourselves and carefully judge what is working and what no longer will, what we have outgrown and what we will wear well. These decisions are often difficult but necessary. They demand a true self-awareness of our own frailties and prejudices and, in the case of associations, often difficult decisions that are necessary for long-term health and well-being. New ideas bring angst and speculation as the future is hard to foresee, the past easy. To do this means constantly reevaluating our ideas and decisions, but once decided, all must join in and work as a unified team if we are to succeed. To question is appropriate, but to divide in order to conquer or arbitrate is not. So before I go, I'd like to say it has truly been my honor to serve as your president and I thank you greatly for your unending support. I look forward to seeing ACE continue its growth over the many years to come. To my family, who had to endure my time in multiple phone calls, meetings, Zoom meetings, and messages, I can only say thank you from the bottom of my heart. I again want to thank each and every one of you for all the hard work and effort you have done this year and all the years in the past and hopefully the future. ACE is worth it. You are worthy of it. As the saying goes, next year somewhere in person again. And now it is my great pleasure to introduce a great friend, compadre, and your next president, Felice Caldarella. Thank you. Thank you, Howard, and greetings to my colleagues. I'm Felice Caldarella and I'm honored to be your next president of the American Association of Clinical Endocrinology. Now I'd like to start by telling you a few things about myself. I'd like to think I have at least one thing in common with each of you other than practicing endocrinology. So here are just a few. First, I'm an immigrant. I was born in Italy. My parents, Francesco and Emilia, who look down from above, immigrated to this country when I was two years old. For all those reasons, immigrants come to the United States. I attended public school, went to college in New York City, and graduated from a state medical school. My practice is in general endocrinology and I see patients five days a week. Beyond my profession, I've been married to my wife, Gina, for 23 years. We have two wonderful children and I'm thankful for the love and support from my family. I've been active with ACE since fellowship. ACE is where I could always go to be educated on endocrine disorders and engage with my colleagues. So now that I've shared a few things about myself, do we have something in common? I hope we do. I believe our community is strengthened by the bonds we share amongst each other, but not only as colleagues, but as friends. I look forward to building these bonds further with you over the next year of my presidency. Let's talk about ACE and the journey we've been on. As Howard said at the beginning of our meeting, ACE was formed over 30 years ago in 1991 by endocrinologists who are motivated as clinicians to improve their well-being as well as the well-being of their patients. We have since grown to be the largest organization of clinical endocrinologists representing both members here in the United States and abroad. We are truly a global organization. We've also transformed our community to be more inclusive to all members of our team who care for people with endocrine disorders. In addition to endocrinologists, we now include nurse practitioners, physician's assistants, pharmacists, and nurses as members. We continue to welcome fellows, residents, and medical students too. The American Association of Clinical Endocrinology is the hub of all those who are motivated, just like all of us, to improve the care of people with endocrine disorders. So we welcome all those who support our mission to elevate clinical endocrinology to improve global health. As our numbers grow, so will our influence and strength. As I begin my presidency, you should expect and I shall work to ensure that we at ACE deliver only the best to help improve the care of our patients. This includes the best information and guidelines, the best journal, endocrine practice to disseminate knowledge, and the best forums to exchange information both in person and online. I will guide our board and our volunteers to focus on the strategic priorities that will truly help us make a difference in our world and in our communities. I am excited about the coming year. You will see me lead an organization that is committed to engaging all of our community members. You will have more opportunities to participate in educational programs. You will see more opportunities for sharing your clinical experience with each other. You will see further development of our disease state networks for those interested in participating. You shall be able to do so based on your interest. The disease state networks are a great way to contribute to ACE. I encourage each of you to participate. In summary, I will work to continue the greatest achievements of the past here at ACE. It also keeps us moving into the future by further strengthening our bonds with all our members and growing our organization to include all those who care for people with endocrine disorders. It is through this strength in our mission to elevate the practice of clinical endocrinology to benefit each individual patient and the health of society that here at ACE we will continue to prosper. I thank you in advance for your support over the next year and I look forward to leading us all into brighter times ahead. Together, let us advance our specialty. Together, let us innovate better care for our patients. Together, let us achieve our mission. Together, we are ACE. Thank you.

Dr. Michael Levine

Chief Emeritus

Endocrinology

Diabetes

Children's Hospital of Philadelphia

vitamin D

precision medicine

vitamin D deficiency

genetics

vitamin D metabolism