Consensus statement from 2nd International Conference on Controversies in Vitamin D.

The 2nd International Conference on Controversies in Vitamin D was held in Monteriggioni (Siena), Italy, September 11-14, 2018. The aim of this meeting was to address ongoing controversies and timely topics in vitamin D research, to review available data related to these topics and controversies, to promote discussion to help resolve lingering issues and ultimately to suggest a research agenda to clarify areas of uncertainty. Several issues from the first conference, held in 2017, were revisited, such as assays used to determine serum 25-hydroxyvitamin D [25(OH)D] concentration, which remains a critical and controversial issue for defining vitamin D status. Definitions of vitamin D nutritional status (i.e. sufficiency, insufficiency and deficiency) were also revisited. New areas were reviewed, including vitamin D threshold values and how they should be defined in the context of specific diseases, sources of vitamin D and risk factors associated with vitamin D deficiency. Non-skeletal aspects related to vitamin D were also discussed, including the reproductive system, neurology, chronic kidney disease and falls. The therapeutic role of vitamin D and findings from recent clinical trials were also addressed. The topics were considered by 3 focus groups and divided into three main areas: 1) "Laboratory": assays and threshold values to define vitamin D status; 2) "Clinical": sources of vitamin D and risk factors and role of vitamin D in non-skeletal disease and 3) "Therapeutics": controversial issues on observational studies and recent randomized controlled trials. In this report, we present a summary of our findings.

Biotin supplementation causes erroneous elevations of results in some commercial serum 25-hydroxyvitamin d (25OHD) assays.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes serum samples globally, on a quarterly basis, to assess participants' performance of specific methods for 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25-(OH)2D). DEQAS occasionally circulates samples containing high levels of substances found in certain clinical situations e.g. 25-OHD2, 24,25-(OH)2D3, hypertriglyceridemia. The increased availability and use of health supplements containing biotin has led to case reports of assay interference in methods utilizing a biotin-streptavidin detection system. In October 2018, DEQAS included a serum sample (545) containing exogenous biotin (concentration =586 µg/L) which was analyzed by a total of 683 laboratories using 35 different methods. The same serum sample (544) without exogenous biotin was also included in the 5-sample set. All methods (760 laboratories) performed satisfactorily on sample 544 giving an All-Laboratory Trimmed Mean = 50.2 ± 6.5 nmol/L (±SD, CV = 12.9 %). The target value for this sample 544 (& 555) was 47.4 nmol/L as determined by Centers for Disease Control and Prevention (CDC) Atlanta, Georgia using their LC-MS/MS reference method. In contrast, #545 containing the exogenous biotin was reported by only 683 laboratories and gave an All-Laboratory Trimmed Mean = 66.8 ± 37.6 nmol/L (±SD, CV = 56.3 %). As expected, LC-MS/MS methods (143 labs) reported similar results for both 544 = 48.9 ± 4.4 nmol/L (±SD) and 545 = 48.3 ± 4.5 nmol/L (±SD) showing that assays involving chromatographic steps are unaffected by the presence of biotin. Several of the antibody-based assays including Abbott Architect, DiaSorin Liaison, Beckman Unicel and Siemens Centaur are also unaffected by the addition of biotin. Two assays, IDS-iSYS and Roche Total 25OHD, both of which use biotin-streptavidin, exhibit biotin interference yielding values with a significant positive bias for 545 of 102.6 nmol/L ± 78.7 nmol/L (±SD) and 517.8 nmol/L ± 209.8 nmol/L (±SD) respectively. Interestingly, the failure to report sample 545 data from 77 laboratories is due solely to those running Roche Total 25OHD or Roche Vitamin D Total II assays. Given the prevalence of the adversely affected assays (25 % of DEQAS users) and the high volume of 25OHD testing, clinicians using these assays should, where possible, only measure 25OHD when patients are off biotin.

External Quality Assessment of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) assays.

The discovery that mutations of the CYP24A1 gene are a cause of idiopathic infantile hypercalcemia (IIH) has revived interest in measuring serum 24,25(OH)2D3. Several studies have also suggested that a high 25-hydroxyvitamin D3(25-OHD3):24,25(OH)2D3 ratio might provide additional diagnostic information in the investigation of vitamin D deficiency. Measurement of 24,25(OH)2D3 is necessarily restricted to laboratories with mass spectrometry methods although cross reactivity of the metabolite in immunoassays for 25-OHD is a potential cause of misleading results. The international External Quality Assessment (EQA) scheme for vitamin D metabolites (DEQAS) was set up in 1989. In 2013 DEQAS became an accuracy based EQA for 25-OHD with 'target values' assigned by the National Institute of Standards and Technology (NIST) Reference Measurement Procedure (RMP). A pilot scheme for serum 24,25(OH)2D3 was started in 2015 and participants were asked to measure the metabolite on each of the 5 samples sent out for 25-OHD. Inter-laboratory agreement was poor but this may reflect methodological differences, in particular different approaches to assay standardization. An important potential contribution to reducing variability among assays was the development by NIST of a 24,25(OH)2D3 RMP and its use in assigning values to SRMs 972a, 2973 and 2971, supported by the NIH Office of Dietary Supplements (ODS) as part of the Vitamin D Standardization Program (VDSP) effort.

Developing vitamin D dietary guidelines and the lack of 25-hydroxyvitamin D assay standardization: The ever-present past.

Unstandardized laboratory measurement of 25-hydroxyvitamin D (25(OH)D) confounds efforts to develop clinical and public health vitamin D guidelines. The Vitamin D Standardization Program (VDSP), an international collaborative effort, was founded in 2010 to correct this problem. Nearly all published vitamin D research is based on unstandardized laboratory 25(OH)D measurements. While it is impossible to standardize all old data, it may be possible to identify a small subset of prior studies critical to guidelines development. Once identified it may be possible to calibrate their 25(OH)D values to the NIST and Ghent University reference measurement procedures using VDSP methods thereby permitting future guidelines to be based on standardized results. We simulated the calibration of a small set of ten clinical trials of vitamin D supplementation on achieved 25(OH)D under minimal sun exposure. These studies were selected because they played a prominent role in setting the 2010 vitamin D dietary reference intakes (DRI). Using random-effects meta-regression analysis, Vitamin D External Quality Assessment (DEQAS) data on assay bias was used to simulate the potential bias due to the lack of assay standardization by calibrating the achieved 25(OH)D levels from those 10 studies to: (1) the largest negative, and (2) the largest positive bias from the DEQAS all laboratory trimmed mean (ALTM) for the appropriate assay and year of analysis. For a usual vitamin D intake of 600IU/day the difference in mean achieved 25(OH)D values for those two options was 20nmol/L. However, without re-calibration of 25(OH)D values it is impossible to know the degree to which any of the current guidelines may have been biased. This approach may help stimulate the search for and standardization of that small subset of key studies and, in the cases where standardization is impossible, to identify areas of urgently needed vitamin D research.

Effectiveness of food fortification in the United States: the case of pellagra.

OBJECTIVES: We evaluated the possible role of niacin fortification of the US food supply and other concurrent influences in eliminating the nutritional deficiency disease pellagra. METHODS: We traced chronological changes in pellagra mortality and morbidity and compared them with the development of federal regulations, state laws, and other national activities pertaining to the fortification of cereal-grain products with niacin and other B vitamins. We also compared these changes with other concurrent changes that would have affected pellagra mortality or morbidity. RESULTS: The results show the difficulty of evaluating the effectiveness of a single public health initiative such as food fortification without controlled experimental trials. Nonetheless, the results provide support for the belief that food fortification played a significant role in the elimination of pellagra in the United States. CONCLUSIONS: Food fortification that is designed to restore amounts of nutrients lost through grain milling was an effective tool in preventing pellagra, a classical nutritional deficiency disease, during the 1930s and 1940s, when food availability and variety were considerably less than are currently found in the United States.

Lack of association between ferritin level and measures of LDL oxidation: the ARIC study. Atherosclerosis Risk in Communities.

Body iron status has been implicated in atherosclerotic cardiovascular disease. The main hypothesis was that high iron status was associated with increased oxidation of LDL. The associations of serum ferritin (a marker of iron status) and dietary iron intake with the susceptibility of LDL to in vitro oxidation (lag phase) and autoantibodies against MDA-modified LDL (two markers of oxidation stress) were examined among 281 men and 192 women with a mean age of 59 years (S.D. = 5) who participated in the Atherosclerosis Risk in Communities (ARIC) Study visit 2 in 1990 through 1992. Lag phase duration and the autoantibodies against MDA-modified LDL were weakly correlated with each other (r = 0.19, P = 0.001 in men; r = 0.15, P = 0.03 in women). In linear regression analysis adjusting for age, field center, blood storage time, and carotid atherosclerosis case-control status, there was no association between ferritin level and the lag-phase, or between ferritin level and autoantibodies against MDA-modified LDL in either sex. Further adjustment for traditional cardiovascular risk factors (smoking, vitamin supplement use, body mass index, LDL cholesterol, hypertension and diabetes) did not alter these null results. Ferritin was significantly and positively correlated with body mass index in both sexes (r = 0.21 among men and r = 0.22 among women) and with the waist-to-hip ratio among women (r = 0.26). In addition, among women, ferritin was positively correlated with orosomucoid (r = 0.24) and with sialic acid (r = 0.19). Dietary iron was not associated with the parameters of LDL oxidation or with ferritin level. These findings do not support a role of body iron stores in promoting oxidation of LDL.

Comparison of the Heart and Estrogen/Progestin Replacement Study (HERS) cohort with women with coronary disease from the National Health and Nutrition Examination Survey III (NHANES III)

BACKGROUND: The Heart and Estrogen/Progestin Replacement Study (HERS) is the first large clinical trial designed to test the efficacy of postmenopausal estrogen/progestin therapy for secondary prevention of coronary heart disease (CHD). To examine the representativeness of the HERS cohort to the general population of postmenopausal women with CHD, we compared the baseline cardiovascular risk factor data from HERS with similar data from women presumed to have CHD from the National Health and Nutrition Examination Survey (NHANES) III. METHODS: Age, race, and cardiovascular disease risk factors were compared in the 2763 postmenopausal women younger than 80 years old, with a uterus, and with documented CHD in HERS versus 145 similarly aged women with clinical or electrocardiographic evidence of CHD from phase I of NHANES III. RESULTS: There were fewer current smokers in HERS (13%) than in the NHANES cohort (21.7%, p = 0.05). Similarly, a history of hypertension was less prevalent in HERS (58.6%) than in the NHANES cohort (69.3%, p = 0.03). Women with fasting triglyceride levels >3.39 mmol/L or fasting glucose levels >16.6 mmol/L were excluded from HERS, resulting in fewer diabetics (22.9% vs 29.5%, p = 0.26) and lower serum triglyceride levels (1.88 mmol/L vs 2.25 mmol/L, p = 0.19) in HERS versus the NHANES cohort. Systolic and diastolic blood pressure, body mass index, physical activity, and total LDL and HDL cholesterol were not significantly different between the two groups. CONCLUSIONS: The HERS cohort had fewer CHD risk factors than women with myocardial infarction or angina in NHANES III, although comparison is hindered by differences in selection criteria. The many women with diabetes and hypertriglyceridemia in the NHANES cohort emphasizes the importance of testing strategies for secondary prevention of CHD in this high-risk subgroup.

Relationship between age and serum vitamin A in children aged 4-11 y.

The association between age and serum vitamin A concentrations in children was examined by using total serum vitamin A values from the second National Health and Nutrition Examination Survey (NHANES II) and serum retinol values for Mexican Americans from the Hispanic HANES. Analyses included multivariate strategies to identify confounders of serum vitamin A. After the effect of the use of vitamin-mineral supplements on total serum vitamin A values was controlled for, the data indicated that younger children (aged 4-5 y) have lower serum vitamin A concentrations than do older children (aged 9-11 y) regardless of whether the measure was total serum vitamin A or serum retinol. This relationship was systematic across the distribution of values and suggested that the difference may be due to normal physiological events. A different interpretive criterion may be needed for younger and older children when serum vitamin A is used to indicate vitamin A status.

Calcium supplementation and bone loss in middle-aged women.

This 4-y study investigated the effects of a calcium carbonate supplement on bone loss in 169 women aged 35-65 y, randomly assigned to placebo and treatment (1500 mg Ca/d) groups in a double-blind design. Bone mineral content (BMC) and width (W) were measured bilaterally on the radius, ulna, and humerus. BMC and BMC/W loss rates were consistently lower in treatment than in control subjects. Loss was significantly reduced in the left and right humerus and the right radius. In premenopausal subjects, only left humerus BMC loss was significantly reduced by Ca supplementation. In postmenopausal treatment subjects, BMC and BMC/W bone loss was reduced in all 12 (bilateral radius, ulna, and humerus) of the bone variables measured, 5 at p less than 0.01 and 2 at p less than 0.05. Ca supplementation counteracted a large portion of the additional bone loss attributable to menopause in this population.

Vitamin-mineral supplement use: association with dietary intake and iron status of adults.

Although vitamin-mineral supplement use is increasing in the United States, few researchers have examined whether supplement users have better nutritional status than do nonusers. Data from 10,515 persons examined in the second National Health and Nutrition Examination Survey (NHANES II) were used to compare mean dietary intakes of several nutrients and food groups, hemoglobin, mean corpuscular volume, transferrin saturation, erythrocyte protoporphyrin, and serum ferritin between regular supplement users and nonusers aged 16 to 74 years. Prevalences of impaired iron status also were compared between user groups. Users consumed more vitamin C and ate fruits and vegetables more frequently than did nonusers in all age/sex groups. No significant differences in mean iron status indicators were observed except in the 65 to 74 year age/sex groups: transferrin saturation among men and mean corpuscular volume, erythrocyte protoporphyrin, and serum ferritin among women. In each case, users had higher values than nonusers in this age group. Prevalences of impaired iron status did not differ between users and nonusers in any age/sex group. In general, iron status was not associated with supplement use.

Comparison of dietary intakes and iron status of vitamin-mineral supplement users and nonusers, aged 1-19 years.

Despite widespread use of supplements, few studies have been conducted to determine if supplement users have better nutritional status. Using data from the second National Health and Nutrition Examination Survey (NHANES II), mean values of five iron status indicators (hemoglobin, mean corpuscular volume, transferrin saturation, erythrocyte protoporphyrin, and serum ferritin) and dietary intakes of several nutrients and food groups were compared between regular supplement users and nonusers aged 1-19 y. Users consumed more vitamin C and fruits and vegetables than nonusers in several age-sex groups. No significant differences in mean Fe status indicator values were observed except for hemoglobin for the 3-4-y olds and serum ferritin for the 5-10-y olds. In both cases, users had higher values than nonusers. In general, Fe status was not associated with supplement use but the reason cannot be identified from this survey.

Effects of intraindividual and interindividual variation in repeated dietary records.

In this study 5,115 daily diet records were collected from 151 women on two randomly selected days per sampling month (29 days) over a two-year period. Yearly estimates of the ratios of intraindividual (sigma w2) and interindividual (sigma b2) variance components were calculated for dietary intake of 15 nutrients and for dietary intake + vitamin/mineral supplements. From one year to the next, corresponding ratios of sigma w2/sigma b2 were nearly identical. Intraindividual variation in dietary intake of all 15 nutrients was greater than interindividual variation (sigma w2/sigma b2 greater than 1). Variance component ratios for highly supplemented nutrients such as iron, vitamin C and B vitamins were less than one (sigma w2/sigma b2 less than 1). Using the ratios of sigma w2/sigma b2, it was calculated that between six and 99 repeated records measuring dietary intake and between two and 31 records measuring total intake may be needed per individual to ensure that the estimate of the population correlation (rho DF) between an individual's "usual" dietary intake or total intake of a dietary risk factor and an individual's mean or usual level of a physiologic risk factor was within 10% of the true population correlation coefficient (rho xy). It was also found that twice as many dietary records per individual were required to estimate the population slope (beta xy) within 10%. These results have serious implications for the design and analysis of prospective nutritional studies.

A two-year dietary survey of middle-aged women: repeated dietary records as a measure of usual intake.

A two-year survey of the dietary consumption and supplement use patterns of adult women was conducted at the University of Wisconsin-Madison. Participants were asked to record their diet on two randomly selected days per month over a two-year period. Each subject filled out an average of 19 records a year, for an overall 71% return rate. Because of the sampling design, it was possible to estimate each woman's "usual" or average daily dietary and total intake (diet plus supplements) of 15 nutrients. Many women were consuming usual amounts of energy, calcium, iron, magnesium, vitamin B-6, and, especially, zinc and total folate that were 30% to 50% below recommended levels. It was also found that the women generally took vitamin/mineral supplements for nutrients already adequately supplied in the diet. Low nutrient intakes may have been due, in part, to underreporting of food intakes.