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1 Supplemental Information SUPPLEMENTARY BACKGROUND REGARDING VITAMIN D AND ITS METABOLITES Vitamin D is the parent compound made in the skin of mammals as cholecalciferol (vitamin D 3 ) or by plants as ergocalciferol (vitamin D 2 ). Vitamin D, whether made in the skin or received as a supplement absorbed from the gut, is taken into the circulation and processed by the liver and other cells into a metabolite called 25-hydroxyvitamin D (25(OH)D; also called calcidiol). 25(OH)D has a longer half-life of 2 to 3 weeks, and therefore, is the indicator of nutritional vitamin D status in the body. It is 25(OH)D that is affected by parathyroid hormone (PTH) and that is converted into the active hormone 1,25(OH) 2 D (also known as calcitriol). 1,2 It is mainly the parent compound (cholecalciferol/ergocalciferol) that is transferred into breast milk with minimal 25(OH)D transferred in breast milk to the recipient breastfeeding infant. 3 5 (For a more complete review of vitamin D metabolism, please refer to articles by Hollis and Wagner 6 8 and Holick. 9 ) METHODS Subjects and Study Sites The study was conducted at the MUSC (latitude N) and the U of R (U of R; latitude N). Approval for this 2-site study was granted by (1) MUSC s Institutional Review Board for Human Subjects HR and the CTRC (Protocol 752) and (2) the U of R s Institutional Review Board for Human Subjects (14460) and the CTRC (Protocol 1129). The study is registered with ClinicalTrial.gov (identifier NCT ). Study Design Lactating Arm of Study This was a randomized, double-blind, comparative effectiveness trial of 3 doses of vitamin D supplementation in lactating mothers and their breastfeeding infants and nonlactating controls. Following written informed consent, mothers were randomized to 1 of 3 vitamin D supplementation regimens substratified by race: Group 1, 400 IU vitamin D 3 /day (0 IU vitamin D 3 placebo and 1 prenatal vitamin containing 400 IU vitamin D 3 ); Group 2, 2400 IU (2000 vitamin D 3 /day and 1 prenatal containing 400 IU vitamin D 3 ); and Group 3, 6400 IU vitamin D 3 /day (6000 IU vitamin D 3 and 1 prenatal vitamin containing 400 IU vitamin D 3 ). The breastfeeding mothers also were provided with a liquid suspension vitamin D supplement to be given to their nursing infants (Bio-D-Mulsion, Biotics Research Corp, Rosenberg, TX). Mothers in each group were instructed to give 1 drop of the suspension per day: those infants in Group 1 received 400 IU vitamin D 3. (The effectiveness of this solution in improving infant vitamin D status was previously demonstrated. 10 ) Infants in Groups 2 and 3 received placebo containing 0 IU vitamin D 3.In addition to the 3 group comparisons, each subject also served as her own control, establishing each mother s vitamin D status (circulating vitamin D and 25(OH)D concentrations) at 1 month then compared monthly at 6 additional time points. The vitamin D status of the infants in the 3 groups was compared at baseline and months 4 and 7. Subject Inclusion Criteria 1. Exclusively/Fully lactating mothers and their breastfeeding singleton infants (receiving no other form of nutrition other than human milk at the time of study entry 11,12 ) within 4 to 6 weeks postpartum living in Charleston, South Carolina, or Rochester, New York, were eligible for inclusion in the study if they planned to continue full breastfeeding for the next 6 months. 11,13 Use of complementary foods starting at 6 months was permitted for all infants as per AAP guidelines and World Health Organization recommendations as long as their sole milk source was maternal breast milk Infants had to be $35 weeks gestation and in good general health at the time of enrollment. Infants receiving a vitamin D supplement before the study were eligible for participation in the study (their results are not included in this analysis). Subject Exclusion Criteria 1. Mothers were not eligible for participation in the study if they had any of the following diagnoses: preexisting type I or II diabetes, hypertension, parathyroid disease, and uncontrolled thyroid disease. Women of twins or multiple births were not eligible to participate in the study. 2. Infants,35 weeks gestation; with a history of.72 hours in the NICU; any inborn error of metabolism; history of congenital anomalies; or a history of consuming.10% SI1

2 of their diet as formula at the time of enrollment were not eligible for participation in the study. Twins and multiples were not eligible for participation in the study. 3. Women who were combination feeders at the time of enrollment (ie, partially breastfeeding and formula-feeding their infants) were not eligible to participate in the study. Those who chose to combination feed after enrollment and before the 4-month study visit exited the study. Block Randomization Mothers were randomized to 1 of the 3 treatment groups using Proc Plan in SAS. This program allowed for the input of the number of strata and estimated sample size. Specifically, a list of random assignments was generated stratified by ethnicity. At the time of enrollment, the study coordinator accessed the randomization Web page from the General Clinical Research Center Web site (later CTRC) developed uniquely for this investigation. Only the Research Pharmacy and the Data Coordinating Center were notified via the computer program that a patient had been newly enrolled and of the group assignment. Investigators, study team, and subject remained blinded to treatment assignment. Study Protocol 1. Visit: Each lactating mother and her breastfeeding infant came to the General Clinical Research Center/CTRC at MUSC or U of R for the baseline visit at 4 to 6 weeks postpartum and for monthly visits through 7 months postpartum. 2. Completion of Questionnaires: Questionnaires regarding sociodemographic information including extent of education in years and level achieved (high school, some college, college graduate, and postbaccalaureate); insurance status (private vs Medicaid/ none); baseline health status (as defined by active, chronic, and previous health problems); and medical history were completed at the first baseline visit. One week before each subsequent visit, each mother was asked to complete questionnaires regarding physical activity, sunlight exposure, and type of clothing worn. An interim health history questionnaire regarding maternal and infant health (type and frequency of acute illnesses such as respiratory, gastrointestinal, and other viral and/or bacterial illnesses; current and past medications and doctor s visits within the past month) was completed at each visit. 3. Infant Gestational Age and Infant Anthropometric Measures at Birth: The infant s gestational age (weeks), birth weight (g), birth length (cm) and birth head circumference were recorded at the first visit and later verified by the medical record. 4. Infant Vitamin D Supplementation: At the time of the initial visit, whether the infant was receiving a vitamin D oral supplement and the number of IU per day were recorded. 5. Breastfeeding History: A detailed infant breastfeeding history was completed by the mother during the week before the scheduled visit and reviewed by the study coordinator at the study visit to determine the current feeding category (fully breastfeeding, combination feeding, or formula feeding). Any supplementation with formula or other foods was noted and quantified. Women who stopped breastfeeding before the fourth visit exited the study; those women who breastfed up to visit 4 but who either stopped breastfeeding or were combination feeding (breastfeeding and formula feeding) continued to be followed for safety reasons, but their feeding type was changed from fully breastfeeding to the current status (data not included in this analysis). 6. Maternal Dietary Intake: Each mother completed a Block 2005 Food Frequency Questionnaire at the second visit to ascertain her generalized eating pattern, with specific calculation of calcium and vitamin D intake (Nutrition Quest, Berkeley, California). This questionnaire was chosen over food diaries or 3-day food records for the following reasons: (1) The questionnaire does not rely on memory as much as other validated methods, (2) its readability is designed for lower literacy populations; and (3) it accurately estimates individual and populationwide average daily intakes of all macronutrients and micronutrients. This tool has been validated and reproduced against other dietary intake tools Each completed Food Frequency Questionnaire form was sent to the processing center (Berkeley, California) for analysis. 7. Monthly Anthropomorphic Measures: a. Maternal: At each study visit, maternal weight was obtained; maternal height was measured at visit 1 and used to determine monthly BMI. Maternal BMI was calculated as follows: weight (kg)/[height (m)]. 2 b. Infant Current Weight, Length, Head Circumference, and Fontanelle Area: Each infant s current weight (kg), length (cm) using a length board, and SI2 HOLLIS et al

3 head circumference (cm) were measured according to standard clinical pediatric practice at the initial baseline visit and each monthly visit. Anterior fontanelle area, (cm 2 : anterior fontanelle length (cm) 3 anterior fontanelle width (cm)) was measured at each visit. 8. Prenatal Vitamins and Vitamin D Tablets: The prenatal vitamins provided to the mothers in the study contained 400 IU vitamin D 3 (United Research Laboratories, Philadelphia, PA). Vitamin D 3 tablets (0, 2000, and 6000) were manufactured by Tishcon Corporation (Westberry, NY), a Good Manufacturing Practice facility that met Food and Drug Administration production guidelines. To achieve the correct dosage of vitamin D supplementation, each mother took 1 prenatal vitamin containing 400 IU vitamin D 3 and 1 vitamin D study tablets daily as follows: 400 IU Group, 1 tablet containing 0 IU vitamin D 3 (placebo), identical in appearance to the other vitamin D tablets; 2400 IU group, 1 tablet containing 2000 IU vitamin D 3 ; and 6400 IU group, 1 tablet containing 6000 IU vitamin D 3. The total vitamin D 3 intake of mothers in Groups 1 and 2 was 400 IU and 6400 IU/day, respectively. 9. Infant Vitamin D Oral Supplement Preparation: Infants in the 400 IU group were given 1 drop of a vitamin D suspension containing 400 IU per drop (Bio-D-Mulsion manufactured by Biotics Research Corp, Rosenberg, TX), previously validated by this study team. 10 Those infants in the 2400 IU and 6400 IU groups received a placebo preparation identical in appearance, taste, and smell to the vitamin D containing preparation also manufactured by Biotics Research Corp. Each mother was instructed on how to properly administer the vitamin preparation and was asked to demonstrate her skill and understanding during her initial study visit. 10. Vitamin D-Free Formula for Emergency Supplementation: During the course of the study, there were occasions when mothers had a procedure or were away from their babies and could not provide breast milk for up to 72 hours. During those times, the baby was given vitamin D free formula manufactured by Mead- Johnson (Evansville, Indiana) to be identical in every other aspect to term 20 kcal/ounce formula but without vitamin D. Supplementation with this formula was recorded by date and time of feeding. Mothers were considered fully breastfeeding if they used less than 10% vitamin D free formula supplementation. Each reconstituted can of vitamin D free formula was 96-ounces and breastfeeding subjects were given only 1 can per month as the maximum amount possible and for emergency use only; reasons vitamin D free formula was given also were recorded. 11. Adherence to Medication Regimen: Adherence to the prescribed vitamin D supplementation regimen of 1 prenatal vitamin and the vitamin D supplement was measured by maternal selfreport and pill counts at each follow-up visit. Adherence to the regimen was defined as the number of pills taken divided by anticipated number of pills to be consumed between study visits. Similarly, adherence with infant study preparation was defined as the starting weight of drops plus dispenser bottle minus weight at study visit divided by anticipated weight of drops to have been consumed for those number of days between study visits. 12. Determination of Skin Pigmentation and Sunlight Exposure Monitoring: Skin pigmentation changes from sunlight exposure were monitored monthly in mother and infant using the SmartProbe 400 (IMS, Inc., Milford, CT), a spectrophotometer device that measures degrees of pigmentation on a continuous scale from 0 to 100, 0 being absolute black and 100 being absolute white. Each mother had pigmentation measurements recorded from her exposed forehead, forearm, underarm, stomach, and anterior knee, with 2 readings averaged and recorded for each site. Each infant had pigmentation measurements recorded from the same sites. Of note, mothers were instructed to use sunscreen if outdoors for.15 minutes. As per AAP guidelines, each mother was advised to avoid direct sunlight exposure of her infant during the first 6 months Collection of Blood Samples: A baseline blood sample was drawn from each mother and infant to measure total circulating 25(OH)D (ng/ml), vitamin D, intact parathyroid hormone (pg/ml), total serum calcium (mg/dl), total serum phosphorus (mg/dl), and total serum creatinine (mg/dl). Maternal blood samples subsequently were drawn monthly while infant blood samples were drawn at visit 4 and visit 7 to monitor safety parameters obtained at baseline. SI3

4 14. Collection of Urine Samples: Maternal and infant urine samples were collected monthly for measurement of urinary calcium to creatinine ratio within 24 hours of collection. 15. Collection of Mother s Milk Samples: Milk samples were obtained from each lactating woman at the time of each study visit was collected to document that mother was in fact still breastfeeding. Milk was stored at 20 C for later analysis of components (results not presented here). Although it was beyond the scope of this study to measure vitamin D compounds in the milk samples given the number of samples and the cost associated with these analyses, the milk antirachitic activity of the breast milk had been measured at these doses and previously had been published by our group. 4,5 Laboratory Measurements Maternal and Infant Baseline Serum Calcium and Phosphorus Studies Using standard methodology and laboratory normative data, serum total calcium (expressed in mg/dl) and inorganic phosphorus (expressed in mg/dl) were measured by MUSC s Clinical Chemistry Laboratory for Charleston for South Carolina subjects and by the University of Rochester s Clinical Chemistry Laboratory for New York subjects. Cross-validation between laboratories was performed in 5% of the samples. Circulating 25(OH)D and Vitamin D (Parent Compound) These compounds were measured in the laboratory of Dr. Bruce Hollis using high performance liquid chromatography and radioimmunoassay techniques as previously described On the basis of clinical laboratory classifications, 26,27 deficiency was defined a priori as total circulating 25(OH)D,50 nmol/l (20 ng/ml) as previously described. 6,25,27,28 The inter- and intraassay coefficient of variation is #10%. Maternal and Infant Circulating intact PTH Concentrations Intact PTH (ipth) was measured by immunoradiometric assay (IRMA) that uses 2 polyclonal antibodies (Diasorin, Stillwater, MN). The first antibody, specific for PTH 39-84, is bound to a solid phase bead. The second antibody is specific for PTH 1-34 and is labeled with 125 I. The adult normal range for ipth in our laboratory is 13 to 54 pg/ml (1.3 to 5.4 pmol/l). Higher vitamin D levels are associated with lower ipth; as vitamin D status improves ipth declines. 29 Outcomes The primary outcome was the change from baseline maternal and infant total circulating 25(OH)D at 4 and 7 months postpartum in exclusively/fully lactating pairs by treatment group, and the secondary outcome was the percent of women and infants by treatment group who achieved a concentration of at least 50 nmol/l (20 ng/ml), meeting the IOM s definition of sufficiency at baseline and at 7 months postpartum. 30 The primary focus of the analysis was to compare the impact of maternal alone versus maternal/infant vitamin D supplementation in a group of women and their fully breastfeeding infants of diverse racial and ethnic backgrounds. The primary comparison made was a general test of group differences in the rate of hypovitaminosis D, regardless of race/ ethnicity by treatment group. Additional secondary outcome measures included to monitor safety were serum intact PTH (ipth), calcium and phosphorus, and urinary calcium/creatinine ratio. The analysis was undertaken using an intention-to-treat approach in which all individuals randomized to 1 group were considered to be within that group throughout the analysis. 31 Exclusive and then full breastfeeding had to be demonstrated at each visit for inclusion in the exclusive/fully breastfeeding analyses. The 3 treatment groups for breastfeeding mothers were compared at entrance into the study to detect potential differences with regard to sociodemographic and baseline clinical characteristics. After stopping the 2400 IU arm due to safety concerns for the infants, beyond the baseline characteristic analyses reported, time-point measures were restricted to the 2 remaining treatment groups 400 IU and 6400 IU. Sample Size Calculation The goal of supplementation, determined a priori, was to achieve a minimum average circulating 25(OH)D of 100 nmol/l (40 ng/ml) in all ethnic groups, a level that represents a clinically relevant concentration in lactating women based on earlier studies. 4,5 For this investigation, we estimated that the circulating 25(OH)D concentrations for Hispanics would be similar to that of African Americans. As such, the sample size needed would be large enough to detect an improvement over baseline to an average 100 nmol/l (40 ng/ml) for all ethnic groups. This represents a 20% improvement for Caucasians and a 75% improvement for African Americans and Hispanics. Thus, based on our findings, a final analytic sample of 15 Caucasians in each supplementation group (paired analysis, a , 2 tailed) would achieve statistical power of 80%. To oversample to accommodate possible attrition and still maintain a balanced design, 21 Caucasian maternal subjects per treatment group were to be enrolled. It was estimated that 4 African Americans/Hispanics in each supplementation group to achieve SI4 HOLLIS et al

5 statistical power of 80%; however, to attain a balanced design and to ensure more stable variances, a final analytic sample of 21 subjects (per treatment group) were enrolled. Accordingly, the study was appropriately powered to detect important differences in final 25 (OH)D versus baseline levels, and to detect dose effects of the final 25(OH)D specific to each ethnic group and each study site. Because the study was developed to assess the change over baseline within each of 3 study original treatment groups and because the sample sizes were estimated based on achieving a minimum of a 20% improvement over baseline, each arm is effectively, and independently, powered to assess achievement of the outcome measure. The loss of the 2400 IU group did not alter the capability of the other arms in assessing their effectiveness because each can be viewed as an independent trial. The blocked stratified randomization ensures balance in ethnicity and breastfeeding status, so the loss of asinglearmdidnotinanywayaffectthe scientific rigor of the other arms. Statistical Analyses Statistical analyses were performed using SAS version 9.3 (SAS, Cary, NC). x 2 analyses were used to test for differences in categorical data. Student s t test analyses were used to test for differences in normally distributed variables. The Wilcoxon rank-sum test was used for analyses involving nonparametric variables. Regression methods (multivariate and logistic) included variables that were significant in bivariate analysis to model 25(OH)D status. Correlation analysis was performed by Spearman s correlation. Relative risk with corresponding confidence intervals was calculated for dichotomous variables, and difference in means with their 95% confidence intervals was used for additional analysis of continuous variables. Significance was set a priori as P,.05. SUPPLEMENTARY RESULTS/ TABLES As shown in Fig 1 CONSORT Diagram of the main manuscript, there were 564 total who were enrolled: 489 lactating women and 86 exclusively formulafeeding women (enrolled as a safety assessment not reported in this article; this subgroup is mentioned in the first exclusion box to the right). An additional 89 women/infant pairs were excluded before randomization for the reasons given in the exclusion box. There were 389 women who were consented, enrolled, and randomized into 1 of the 3 treatment groups; however, because the 2400 IU group was ended by the DSMC, only the 400 and 6400 IU groups are reported in the Results section (n 5 334). Of the 334 women who came to the first study visit, 216 (64.7%) continued exclusively breastfeeding to visit 1; 148 (44.3%) continued exclusively breastfeeding through visit 4; and 95 (28.4%) continued to exclusively breastfeed (with complementary foods) through visit 7. Additional specific safety data regarding differences between V1 and V4 mothers and infants and the V1 and V7 mothers and infants by treatment group (400 vs 6400 IU groups) are found in Supplementary Tables 3 through 6. Groups were compared by serum total calcium, creatinine and urinary calcium/creatinine, parathyroid hormone (PTH), and maternal vitamin D (parent compound, cholecalciferol, and ergocalciferol). Two of the infants in the 6400 IU group met the definition of vitamin D deficiency with a 25(OH)D level,20 ng/ml at 4 months. Documentation of adherence was limited to maternal report, pill count, and weighing of the liquid vitamin D dispenser at each visit. In these 2 cases, weighing of the liquid vitamin container suggests noncompliance as the etiology of the infants deficiency states. This could not be otherwise verified. Because the data were analyzed on an intent-to-treat basis, the infants were included in the analyses. Analyses of the parent compound vitamin D concentration by racial/ethnic group were confined to Hispanic and White women because there were insufficient numbers of black women on whom data were available. For Hispanic and White women, although there were no differences in vitamin D concentrations at baseline for treatment group (P 5.17 and 0.16, respectively), differences were apparent at V4. Specifically, for Hispanic women at V4, the mean vitamin D 6 SD concentration was nmol/l (range ) for women in the 400 IU group (n 5 14) compared with nmol/l for women in the 6400 IU group (n 5 8; P ). For white women at V4, the mean vitamin D 6 SD concentration for the 400 IU group (n 5 37) was nmol/l (range ; n 5 37) compared with (range ) in the 6400 IU group (n 5 41; P,.0001). In the model predicting maternal 25 (OH)D in exclusively breastfeeding mothers at V4, controlling for treatment, race/ethnicity, site, and BMI.30, there were certain independent predictors: treatment had the most significant effect on women in the 6400 IU group with a parameter increase in 25(OH)D concentration of nmol/l compared with the 400 IU group (P,.0001). With regard to race/ethnicity, compared with white women, black women had nmol/l (P,.0001) and Hispanic women nmol/l (P,.0001) lower 25(OH)D concentrations. BMI.30 was associated with nmol/l lower 25(OH)D than those women who had a BMI #30 (P ). SI5

6 In predicting the difference in 25(OH)D concentration between V1 and V4, only treatment remained significant in the model with 6400 IU/day associated with an increased 25(OH)D by nmol/l (P,.0001). In the model predicting maternal 25(OH)D at V7, the same independent predictors at V4 remained significant with site (MUSC) showing an increase in 25(OH)D by nmol/l (P 5.038). At V7, the parameter estimate increased to nmol/l (P,.0001) in the 6400 IU group compared with the 400 IU group; being Black the loss estimate increased to nmol/l (P ) and being Hispanic the loss estimate increased to nmol/l (P ); BMI.30 loss increased slightly to nmol/l (P 5.028). In the modeling looking at the change in 25(OH)D between V1 and V7, only treatment with 6400 IU remained significant ( nmol/l; P,.0001). SUPPLEMENTARY DISCUSSION With regard to pediatricians versus obstetricians care of the breastfeeding dyad, there is concern that pediatricians could not recommend a higher dose of vitamin D for the lactating mother. Competency regarding the care of the breastfeeding dyad is part of the American Board of Pediatrics goals for residency programs and recertification. It was the AAP that put forth the recommendation for universal infant vitamin D supplementation in that was reissued in It is the pediatrician who typically supports the breastfeeding mother-infant dyad, 14 who prescribes the vitamin D supplement for the infant, and who must be aware of alternatives to infant supplementation, in this case, maternal supplementation. Dissemination of this information to the obstetrical community also is essential but ultimately it is the pediatrician who makes the final recommendation about the breastfeeding dyad. Although exact numbers of infantile rickets is not known due to underreporting and the lack of mandated reportingby state health departments across the United States, there is evidence to suggest that some young infants who present with multiple fractures thought to be due to child abuse may in fact be the result of vitamin D deficiency and rickets The IOM has recommended that infants have a total circulating 25(OH) D level of 50 nmol/l (20 ng/ml), the level recommended for other age groups. Infants with 25(OH)D levels,37 nmol/l (12 ng/ml) have a greater risk of developing rickets. Between the development of rickets and sufficiency, there may be other health effects such as immune compromise with a higher risk of respiratory syncytial virus in the first year of life 39,40 and neurodevelopmental sequelae. 41 How does vitamin D supplementation to the lactating women affect the nutritional 25(OH)D status of her nursing infant? Limited small studies exist on this topic. 5,42,43 Ala-Houhala et al demonstrated a limited increase in the nursing infants circulating 25(OH)D levels by supplementing lactating mothers with 1000 to 2000 IU/d vitamin D 3. 42,43 Our group, using maternal supplementation of 2000 and 4000 IU/d vitamin D 2, had limited success at raising infant circulating 25(OH)D. 4 A recent vitamin D supplementation RCT during lactation confirmed our findings that maternal supplementation of 2000 IU/d does not sustain the breastfeeding infant. 44 Our current study confirms this finding that 2400 IU vitamin D 3 /day to the nursing mothers will not adequately support her nursing infant s vitamin D status. Our final pilot study before the present RCT, however, provided remarkable results. 5 By supplementing lactating mothers with 6400 IU/d vitamin D 3 for a period of 6 months we dramatically raised the milk vitamin D content and nursing infant circulating 25(OH)D concentrations: the results are consistent in both trials. The strengths of this 2-site study are that it was conducted at 2 distinct latitudes with strong racial/ethnic diversity such that the results can be applied to a wide-range of breastfeeding mothers and their infants. Additional strengths of the study are that it was conducted as a RCT to assess the effectiveness of 3 treatments and included nonlactating women as controls to ensure safety of the higher treatment dose of 6400 IU vitamin D 3 /day. Monthly maternal and infant laboratory measures further ensured the safety of the higher dose treatment groups. Another strength of the study was our ability to account for the contribution of sunlight exposure using an objective measure of skin pigmentation changes across the study period for each subject. The interim analysis allowed identification early-on of the inferiority of the 2400 IU vitamin D dosing in achieving vitamin D sufficiency in the fully breastfeeding infant, which led to discontinuation of that study arm. Limitations of the study as discussed in the main article include the high attrition rate of women who were initially enrolled in the study but who stopped breastfeeding or became combination feeders, excluding them from continued participation in the lactation arm of the study. For safety reasons, these women were followed, and their safety parameters did not differ by treatment group (data not shown but previously presented). 45 The number of women in this study who stopped exclusively breastfeeding mirrors the national rates as measured by the Centers for Disease Control and Prevention. 46 SI6 HOLLIS et al

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Washington, DC: National Academy Press; Appelgren KE, Nietert PJ, Hulsey TC, Hollis BW, Wagner CL. Analyzing adherence to prenatal supplement: does pill count measure up? Int J Endocrinol. 2010;2010: Gartner LM, Greer FR; Section on Breastfeeding and Committee on Nutrition. American Academy of Pediatrics. Prevention of rickets and vitamin D deficiency: new guidelines for vitamin D intake. Pediatrics. 2003;111(4 pt 1): Wagner CL, Greer FR; American Academy of Pediatrics Section on Breastfeeding; American Academy of Pediatrics Committee on Nutrition. Prevention of rickets and vitamin D deficiency in infants, children, and adolescents. Pediatrics. 2008;122(5): Ayoub D, Plunkett J, Keller KA, Barnes PD. Are Paterson s critics too biased to recognize rickets? Acta Paediatr. 2010;99(9): Hyman CJ, Ayoub D, Miller M. Response to Taylor et al: comments on making the diagnosis of rickets in asymptomatic young children. Clin Pediatr (Phila). 2011;50(5): Ayoub D. Fractures: abuse or rickets? Radiology. 2012;264(2): , author reply Ayoub D. Limitations of radiology in rickets. Pediatr Dev Pathol. 2013;16: Paterson CR, Ayoub D. Congenital rickets due to vitamin D deficiency in the mothers. Clin Nutr. 2014;S (14) Hansdottir S, Monick MM, Lovan N, Powers L, Gerke A, Hunninghake GW. Vitamin D decreases respiratory syncytial virus induction of NF-kappaB-linked chemokines and cytokines in airway epithelium while maintaining the antiviral state. J Immunol. 2010;184(2): Belderbos ME, Houben ML, Wilbrink B, et al. Cord blood vitamin D deficiency is SI7

8 associated with respiratory syncytial virus bronchiolitis. Pediatrics. 2011;127(6): e1513 e Whitehouse AJ, Holt BJ, Serralha M, Holt PG, Kusel MM, Hart PH. Maternal serum vitamin D levels during pregnancy and offspring neurocognitive development. Pediatrics. 2012;129(3): Ala-Houhala M. 25-Hydroxyvitamin D levels during breast-feeding with or without maternal or infantile supplementation of vitamin D. J Pediatr Gastroenterol Nutr. 1985;4(2): Ala-Houhala M, Koskinen T, Terho A, Koivula T, Visakorpi J. Maternal compared with infant vitamin D supplementation. Arch Dis Child. 1986;61(12): Czech-Kowalska J, Latka-Grot J, Bulsiewicz D, et al. Impact of vitamin D supplementation during lactation on vitamin D status and body composition of motherinfant pairs: a MAVID randomized controlled trial. PLoS One. 2014;9(9): e Wagner C, Howard C, Ebeling M, et al. Safety of continued maternal vitamin D supplementation in formula feeding mothers or those who stop breastfeeding: results of NICHD RCT. Presented at the Pediatric Academic Societies meeting; 2015; San Diego, CA 46. Division of Nutrition, Physical Activity, and Obesity, National Center for Chronic Disease Prevention and Health Promotion. Breastfeeding Among U.S. Children Born , CDC National Immunization Survey. Available at: breastfeeding/data. Accessed August 6, 2015 SUPPLEMENTAL TABLE 3 Laboratory Parameters of Fully Breastfeeding Mothers Through V4 Maternal Laboratory Values V1 V4 400 IU Group 6400 IU Group P 400 IU Group 6400 IU Group P 25(OH)D (nmol/l) n * 74 74,.0001* Mean 6 SD Range (OH)D,50 nmol/l, n (%) 7 (9.5%) 5 (6.8%).55 7 (9.5%) 1 (1.4%).063 a Relative risk 400 IU: 6400 IU b 7.0 Vitamin D 3 (nmol/l) n ,.0001* Mean 6 SD Range Serum calcium (mmol/l) n Mean 6 SD Range Serum creatinine (mmol/l) n Mean 6 SD Range Urinary Ca (mmol/l)/cr (mmol/l) ratio n Mean 6 SD Range Phosphorus (mmol/l) n Mean 6 SD Range Intact PTH (pmol/l) n Mean 6 SD Range * P,.05. a Fisher s exact test. b Relative risk defined as 25(OH)D,50 nmol/l at V4: comparison of 400 IU Group to 6400 IU Group. SI8 HOLLIS et al

9 SUPPLEMENTAL TABLE 4 Laboratory Parameters of Fully Breastfeeding Mothers Who Completed All 7 Study Visits Maternal Laboratory Values V1 V4 V7 400 IU 6400 IU P 400 IU 6400 IU P 400 IU 6400 IU P 25(OH)D (nmol/l) n , ,.0001* Mean 6 SD Range (OH)D,50 nmol/l, n (%) 4 (8.5%) 3 (9.5%).71 a 5 (10.6%) 1 (2.1%).11 a 6 (12.8%) 1 (2.1%).059 Relative risk: 400 IU: 6400 IU b Vitamin D 3 (nmmol/l) n , ,.0001* Mean 6 SD Range Serum calcium (mmol/l) n Mean 6 SD Range Serum creatinine (mmol/l) n Mean 6 SD Range Urinary Ca (mmol/l)/cr (mmol/l) ratio n Mean 6 SD Range Phosphorus (mmol/l) n * Mean 6 SD Range Intact PTH (pmol/l) n * Mean 6 SD Range * P,.05. a Relative risk defined as 25(OH)D,50 nmol/l at V4 and V7: comparison of 400 IU group to 6400 IU group. b Fisher s exact test. SI9

10 SUPPLEMENTAL TABLE 5 Laboratory Parameters of Fully Breastfeeding Infants Through V4 Infant Laboratory Values V1 V4 400 IU Group 6400 IU Group P 400 IU Group 6400 IU Group P 25(OH)D (nmol/l) n Mean 6 SD Range (OH)D,50 nmol/l, n (%) 58 (78.4%) 53 (71.6%).34 9 (12.2%) 2 (2.7%).056 a Relative risk b 4.5 Serum calcium (mmol/l) n Mean 6 SD Range Serum creatinine (mmol/l) n Mean 6 SD Range Urinary Ca (mmol/l)/cr (mmol/l) ratio n Mean 6 SD Range Phosphorus (mmol/l) n Mean 6 SD Range Intact PTH (pmol/l) n Mean 6 SD Range a Fisher s exact test. b Relative risk defined as 25(OH)D,50 nmol/l at V4: comparison of 400 IU group to 6400 IU group breastfeeding infants. SI10 HOLLIS et al

11 SUPPLEMENTAL TABLE 6 Laboratory Parameters of Fully Breastfeeding Infants Who Completed All 7 Study Visits Infant Laboratory Values V1 V4 V7 400 IU 6400 IU P 400 IU 6400 IU P 400 IU 6400 IU P 25(OH)D (nmol/l) n Mean 6 SD Range (OH)D,50 nmol/l, n (%) 37 (78.7%) 36 (75.0%) (4.3%) 2 (4.2%) 1.0 a Relative risk 1.0 Serum calcium (mmol/l) n Mean 6 SD Range Serum creatinine (mmol/l) n Mean 6 SD Range Urinary Ca (mmol/l)/cr (mmol/l) ratio n Mean 6 SD Range Phosphorus (mmol/l) n Mean 6 SD Range Intact PTH (pmol/l) n Mean 6 SD Range a by Fisher s Exact Test SI11