The

n e w e ng l a n d j o u r na l

of

m e dic i n e

c or r e sp ondence

Vitamin D–Binding Protein and Vitamin D in Blacks and Whites To the Editor: Current assays for vitamin D sufficiency measure total circulating 25-hydroxyvitamin D. However, Powe et al. (Nov. 21 issue)1 conclude that measurement of 25-hydroxyvitamin D that is not bound to the vitamin D–binding protein, which they refer to as bioavailable 25-hydroxyvitamin D, provides a better assessment of sufficiency. They report that the correlation between levels of bioavailable 25-hydroxy­ vitamin D and levels of parathyroid hormone is stronger than the correlation between levels of total circulating 25-hydroxyvitamin D and levels of parathyroid hormone when evaluated across races. However, they fail to reconcile their findings and the rationale underlying their conclusion with a critical role of the complex of 25-hydroxy­ vitamin D and vitamin D–binding protein. This complex is taken up by renal proximal tubule epithelial cells through receptor-mediated endocytosis. The 25-hydroxyvitamin D component of the endocytosed complex then becomes the major precursor for circulating 1,25-dihydroxy­ vitamin D, the active steroid hormone form of vitamin D that is important in the regulation of parathyroid hormone levels.2,3 Thus, in the regulation of parathyroid hormone, the “bioavailable” this week’s letters

878

878

Vitamin D–Binding Protein and Vitamin D in Blacks and Whites

881

Nut Consumption and Mortality

882

Laryngeal Mask Airway in Medical Emergencies

884

Dead Man Walking

885

Two Cases of Invasive Vancomycin-Resistant Group B Streptococcus Infection

form of 25-hydroxyvitamin D is, in fact, the form that is bound to the vitamin D–binding protein. Steven J. Weintraub, M.D. St. Louis Veterans Affairs Medical Center St. Louis, MO [email protected] No potential conflict of interest relevant to this letter was reported. 1. Powe CE, Evans MK, Wenger J, et al. Vitamin D–binding

protein and vitamin D status of black Americans and white Americans. N Engl J Med 2013;369:1991-2000. 2. Nykjaer A, Dragun D, Walther D, et al. An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3. Cell 1999;96:507-15. 3. Leheste JR, Melsen F, Wellner M, et al. Hypocalcemia and osteopathy in mice with kidney-specific megalin gene defect. FASEB J 2003;17:247-9. DOI: 10.1056/NEJMc1315850

To the Editor: Powe et al. proposed that the most appropriate cross-racial marker of vitamin D status may be bioavailable 25-hydroxyvitamin D. This concept of “bioavailability” is analogous to other lipophilic hormones, such as testosterone, in that both free and albumin-bound ligands are available to exert biologic functions. However, the method used to determine bioavailable 25-hydroxy­ vitamin D is, in my view, questionable. The authors adapted the Vermeulen equation, initially developed to calculate free and bioavailable testosterone.1 Estimated concentrations of bioavailable 25-hydroxyvitamin D in the study were compared with concentrations measured with the use of an analogue radioligand assay (Pearson’s r = 0.81 and 0.90). The assay is based on a competitive principle whereby a radiolabeled analogue competes with endogenous bioavailable 25-hydroxyvitamin D for a limited number of vitamin D–binding proteins immobilized on a solid surface. Unfortunately, analogue immunoassay methods for free and bioavailable testosterone are no longer recommended, because es-

n engl j med 370;9 nejm.org february 27, 2014

The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF GEORGIA on August 10, 2015. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.

correspondence

timates by this approach reflect total hormone levels more closely than the free fraction.2-4 The authors should validate their estimation of free 25-hydroxyvitamin D, bioavailable 25-hydroxy­ vitamin D, or both with direct measurement by means of gas chromatography–mass spectrometry or liquid chromatography–mass spectrometry after ultrafiltration or equilibrium dialysis. Yu Chen, M.D., Ph.D. Horizon Health Network Fredericton, NB, Canada [email protected] No potential conflict of interest relevant to this letter was reported. 1. Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation

of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab 1999;84:3666-72. 2. Rosner W, Auchus RJ, Azziz R, Sluss PM, Raff H. Position statement: utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement. J Clin Endocrinol Metab 2007;92:405-13. 3. Fritz KS, McKean AJ, Nelson JC, Wilcox RB. Analog-based free testosterone test results linked to total testosterone concentrations, not free testosterone concentrations. Clin Chem 2008; 54:512-6. 4. Chen Y, Yazdanpanah M, Wang XY, Hoffman BR, Diamandis EP, Wong PY. Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry. Clin Biochem 2010;43:490-6. DOI: 10.1056/NEJMc1315850

polyclonal vitamin D–binding protein antibodies generated against mixed vitamin D–binding protein subtypes.3 Contrary to the results of Powe et al., serum vitamin D–binding protein is generally lower in Gc2 homozygotes than in persons with other Gc genotypes.4 Furthermore, differences in vitamin D–binding protein affinity owing to Gc genotype may be smaller than those used in the formula to calculate bioavailable 25-hydroxyvitamin D levels, because in-depth analysis under different conditions (buffers, pH, and temperature) showed no substantial differences.5 In our view, black Americans, as compared with white Americans, have lower levels of not only total 25-hydroxyvitamin D but also free or bioavailable 25-hydroxyvitamin D. Roger Bouillon, M.D., Ph.D. Katholieke Universiteit Leuven Leuven, Belgium [email protected]

Kerry Jones, Ph.D. Inez Schoenmakers, Ph.D. Medical Research Council Human Nutrition Research Cambridge, United Kingdom No potential conflict of interest relevant to this letter was reported. 1. Winters SJ, Chennubhatla R, Wang C, Miller JJ. Influence of

To the Editor: Powe et al. report that participants homozygous for the Gc1F phenotype of vitamin D–binding protein (>90% of black homozygotes), as compared with participants homozygous for the Gc1S or Gc2 phenotype (>90% of white homozygotes), have lower plasma levels of vitamin D–binding protein and similar levels of bioavailable 25-hydroxyvitamin D, despite lower levels of total 25-hydroxyvitamin D. However, the two monoclonal antibodies in the R&D Systems vitamin D–binding protein assay used in the study may not have equal affinity for all genotypes. As in the article by Powe et al., we found that plasma levels of vitamin D–binding protein measured with the R&D Systems assay were approximately 50% lower in Gambians (blacks only) than in participants from the United Kingdom (whites only) (unpublished data). However, in agreement with Winters et al.,1 we found similar vitamin D– binding protein concentrations in black (Congolese) and white (Belgian) men2 and in black Gambians (approximately 80% Gc1F homozygotes) and white participants from the United Kingdom (approximately 80% Gc1S or Gc2 homozygotes) (unpublished data) with an assay using

obesity on vitamin D-binding protein and 25-hydroxy vitamin D levels in African American and white women. Metabolism 2009; 58:438-42. 2. M’Buyamba-Kabangu JR, Fagard R, Lijnen P, Bouillon R, Lissens W, Amery A. Calcium, vitamin D-endocrine system, and parathyroid hormone in black and white males. Calcif Tissue Int 1987;41:70-4. 3. Bouillon R, van Baelen H, de Moor P. The measurement of the vitamin D-binding protein in human serum. J Clin Endocrinol Metab 1977;45:225-31. 4. Lauridsen AL, Vestergaard P, Nexo E. Mean serum concentration of vitamin D-binding protein (Gc globulin) is related to the Gc phenotype in women. Clin Chem 2001;47:753-6. 5. Bouillon R, van Baelen H, de Moor P. Comparative study of the affinity of the serum vitamin D-binding protein. J Steroid Biochem 1980;13:1029-34. DOI: 10.1056/NEJMc1315850

To the Editor: Powe et al. reported that black participants and white participants have different vitamin D requirements on the basis of the circulating concentration of vitamin D–binding protein. The authors used an assay for vitamin D–binding protein that is untested for the key measurement in their study. This assay is based on a primary monoclonal antibody produced against a single peptide fragment of the human vitamin D–binding protein. Standard vitamin D– binding protein assays are based on polyclonal

n engl j med 370;9 nejm.org february 27, 2014

The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF GEORGIA on August 10, 2015. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.

879

The

n e w e ng l a n d j o u r na l

antibodies against an intact vitamin D–binding protein.1-3 The problem is that the monoclonalantibody assay gives lower values in blacks who have primarily the Gc1F variant of vitamin D– binding protein. The monoclonal antibody discriminates between the Gc1F and Gc1S variants to provide artificially low values for total vitamin D–binding protein in blacks, thus bringing the conclusion of this study into question. As shown in Figure S4 in the Supplementary Appendix (available with the full text of the article at NEJM.org), “calculated” concentrations of bioavailable 25-hydroxyvitamin D correlate well with physically measured concentrations in participants homozygous for either the Gc1F or Gc1S variant, but the slopes of the relationships are very different. Specifically, for any given physically measured level of bioavailable 25-hydroxy­ vitamin D, the authors are overestimating bioavailable 25-hydroxyvitamin D by 2 to 2.5 times owing to underestimation of vitamin D–binding protein in blacks. Bruce W. Hollis, Ph.D. Medical University of South Carolina Charleston, SC [email protected]

Daniel D. Bikle, M.D., Ph.D. University of California, San Francisco San Francisco, CA No potential conflict of interest relevant to this letter was reported. 1. Bikle DD, Halloran BP, Gee E, Ryzen E, Haddad JG. Free

25-hydroxyvitamin D levels are normal in subjects with liver disease and reduced total 25-hydroxyvitamin D levels. J Clin Invest 1986;78:748-52. 2. Bouillon R, van Baelen H, de Moor P. The measurement of the vitamin D-binding protein in human serum. J Clin Endocrinol Metab 1977;45:225-31. 3. Walsh PG, Haddad JG. “Rocket” immunoelectrophoresis assay of vitamin D-binding protein (Gc globulin) in human serum. Clin Chem 1982;28:1781-3. DOI: 10.1056/NEJMc1315850

The authors reply: Weintraub notes that our findings may not reconcile with previous data suggesting that vitamin D–binding protein– bound 25-hydroxyvitamin D is the primary precursor for 1,25-dihydroxyvitamin D. Megalin is a vitamin D–binding protein receptor and takes up vitamin D–binding protein–bound 25-hydroxy­ vitamin D in the proximal renal tubules. Megalin knockout mice are susceptible to bone disease and signs of vitamin D deficiency.1 In contrast, vitamin D–binding protein knockout mice do not have signs of deficiency on a vitamin D–replete diet, a finding that indicates that non–vitamin D– 880

of

m e dic i n e

binding protein–bound 25-hydroxyvitamin D may be sufficient.2 Megalin also binds albumin; thus, albumin-bound 25-hydroxyvitamin D (bioavailable 25-hydroxyvitamin D) may be a relevant analyte converted to 1,25-dihydroxyvitamin D. Chen writes that we should validate the assay used for direct measurement of bioavailable vitamin D because previous studies have found problems with analogue immunoassay methods for directly measuring free testosterone. These issues may not be applicable to our assay method, because our assay is different in its calibrator and affinity-binding approach. We agree that validation of direct assays for bioavailable 25-hydroxy­ vitamin D would strengthen our findings. Bouillon et al. and Hollis and Bikle raise concerns regarding the use of the monoclonal antibody–based vitamin D–binding protein assay, because it may have differential immunoreactivity against variant vitamin D–binding proteins. We were unable to directly address this issue, because genotype-specific standards were not available. Previous research using polyclonal immunoassays did not reveal racial differences in levels of circulating vitamin D–binding protein.3 However, polyclonal antibodies raised against vitamin D– binding protein may cross-react with other proteins.4 Consistent with this observation, when we spiked serum with pooled vitamin D–binding protein, we recovered 91 to 103% of the added vitamin D–binding protein using the R&D Systems kit (monoclonal format) but recovered 171 to 176% using the ALPCO Diagnostics kit (polyclonal format). Furthermore, vitamin D–binding protein levels determined with the use of the polyclonal assay did not correlate with our directly measured results (Fig. S6 in the Supplementary Appendix). Hollis and Bikle suggest that an underestimation of vitamin D–binding protein concentrations in blacks could explain the different slopes observed for Gc1S homozygotes and Gc1F homozygotes (Fig. S4 in the Supplementary Appendix). In our direct assay, vitamin D–binding protein enriched in Gc1S was used as a calibrator and adsorbed affinity-binding reagent. This assay format may result in asymmetric competition for binding between the Gc1S vitamin D–binding protein adsorbed to the plate and Gc1F vitamin D–binding protein in participants’ serum. Buffer conditions also may have differentially influenced the binding affinities of the vitamin D– binding protein variants and thus altered slopes.

n engl j med 370;9 nejm.org february 27, 2014

The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF GEORGIA on August 10, 2015. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.

correspondence

To compare absolute levels of bioavailable 25hydroxyvitamin D in participants of differing genotypes, genotype-specific calibrators are needed. Importantly, the significant association that we observed between total 25-hydroxyvitamin D levels and vitamin D–binding protein genetic polymorphisms was independent of vitamin D–binding protein levels. Camille E. Powe, M.D. Brigham and Women’s Hospital Boston, MA

S. Ananth Karumanchi, M.D. Beth Israel Deaconess Medical Center Boston, MA

Since publication of their article, the authors report no further potential conflict of interest. 1. Nykjaer A, Dragun D, Walther D, et al. An endocytic pathway

essential for renal uptake and activation of the steroid 25-(OH) vitamin D3. Cell 1999;96:507-15. 2. Safadi FF, Thornton P, Magiera H, et al. Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein. J Clin Invest 1999;103:239-51. 3. Winters SJ, Chennubhatla R, Wang C, Miller JJ. Influence of obesity on vitamin D-binding protein and 25-hydroxy vitamin D levels in African American and white women. Metabolism 2009; 58:438-42. 4. Christiansen M, Jørgensen CS, Laursen I, Hirschberg D, Hojrup P, Houen G. Protein chemical characterization of Gc globulin (vitamin D-binding protein) isoforms; Gc-1f, Gc-1s and Gc-2. Biochim Biophys Acta 2007;1774:481-92. DOI: 10.1056/NEJMc1315850

Ravi Thadhani, M.D., M.P.H. Massachusetts General Hospital Boston, MA

Nut Consumption and Mortality To the Editor: The study by Bao et al. (Nov. 21 issue)1 documenting the inverse association between nut consumption and total and cause-specific mortality was déjà vu for me. More than 20 years ago, I published a letter questioning whether it was nuts or magnesium that provided protection against the risk of coronary heart disease in the Adventist Health Study.2 It is well documented that nuts have a higher magnesium nutrient density than essentially any other food.3 The findings by Bao et al. are similar to those for magnesium intake or serum magnesium level with regard to several cause-specific mortalities. A recent review documented an inverse association between the risk of cardiovascular disease and circulating and dietary magnesium.4 An inverse relationship between diabetes and magnesium has been documented in several studies.5 Thus, if the data on estimated magnesium intake or serum magnesium levels are available for this study, I recommend an analysis of the data based on these variables. For the future, I recommend that any study evaluating the effect of nut consumption on health and disease include an estimate of magnesium intake, serum magnesium levels, or both. Ronald J. Elin, M.D., Ph.D. University of Louisville Louisville, KY [email protected] No potential conflict of interest relevant to this letter was reported.

1. Bao Y, Han J, Hu FB, et al. Association of nut consumption

with total and cause-specific mortality. N Engl J Med 2013;369: 2001-11. 2. Elin RJ, Hosseini JM. Is the magnesium content of nuts a factor for coronary heart disease? Arch Intern Med 1993;153:779-80. 3. Elin RJ. Magnesium metabolism in health and disease. Dis Mon 1988;34:161-218. 4. Del Gobbo LC, Imamura F, Wu JH, de Oliveira Otto MC, Chiuve SE, Mozaffarian D. Circulating and dietary magnesium and risk of cardiovascular disease: a systematic review and metaanalysis of prospective studies. Am J Clin Nutr 2013;98:160-73. 5. Dong JY, Xun P, He K, Qin LQ. Magnesium intake and risk of type 2 diabetes: meta-analysis of prospective cohort studies. Diabetes Care 2011;34:2116-22. DOI: 10.1056/NEJMc1315777

To the Editor: Drinking alcohol while consuming an assortment of (often mixed) nuts is a widely popular social habit that represents an entire spectrum of socioeconomic factors and related health behaviors, which by themselves could be associated with mortality outcomes.1 Bao et al. did not take into account the substantial linear correlation between the quantities of alcohol intake and the frequency of nut consumption in the analyses of the pooled and separate cohorts. The relative differences in the mean alcohol intake between participants who consumed nuts seven or more times per week and those who never consumed nuts were 88% in the pooled cohort (Table 1 of the article), 59% in the cohort of women (Table S3 in the Supplementary Appendix, available with the full text of the article at NEJM.org), and 45% in the cohort of men (Table S3 in the Supplementary Appendix). Such

n engl j med 370;9 nejm.org february 27, 2014

The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF GEORGIA on August 10, 2015. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.

881

Vitamin D-binding protein and vitamin D in blacks and whites.

Vitamin D-binding protein and vitamin D in blacks and whites. - PDF Download Free
408KB Sizes 2 Downloads 3 Views