Calcium isotope fractionation by osteoblasts and osteoclasts, across endothelial and epithelial cell barriers, and with binding to proteins

Abstract

Timely and accurate diagnosis of osteoporosis is essential for adequate therapy. Calcium isotope ratio (δ44/42Ca) determination has been suggested as a sensitive, non-invasive and radiation-free biomarker for the diagnosis of osteoporosis, reflecting bone calcium balance. The quantitative diagnostic is based on the calculation of the δ44/42Ca difference between blood, urine and bone. The underlying cellular processes, however, have not been studied systematically. We quantified calcium transport and δ44/42Ca fractionation during in-vitro bone formation and resorption by osteoblasts and osteoclasts and across renal proximal tubular epithelial cells (HK-2), endothelial cells (HUVEC) and enterocytes (Caco-2) in transwell systems, and determined transepithelial electrical resistance characteristics. δ44/42Ca fractionation was furthermore quantified with calcium binding to albumin and collagen. Calcified matrix formed by osteoblasts was isotopically lighter than culture medium by -0.27 ± 0.03‰ within 5 days, while a consistent effect of activated osteoclasts on δ44/42Ca could not be demonstrated. A transient increase in δ44/42Ca in the apical compartment by 0.26‰ occured across HK-2 cells, while δ44/42Ca fractionation was small across the HUVEC barrier, and absent with Caco-2 enterocytes, and with binding of calcium to albumin and collagen. In conclusion, δ44/42Ca fractionation follows similar universal principles as during inorganic mineral precipitation; osteoblast activity results in δ44/42Ca fractionation. δ44/42Ca fractionation also occurs across the proximal tubular cell barrier and needs to be considered for in-vivo bone mineralization modelling. In contrast, the effect of calcium transport across endothelial and enterocyte barriers on blood δ44/42Ca should be low and is absent with physiochemical binding of calcium to proteins.

Nutritional Calcium Supply Dependent Calcium Balance, Bone Calcification and Calcium Isotope Ratios in Rats

“…The δ 44/42 Ca gradient between bone and serum (δ 44/42 Ca Bone -δ 44/42 Ca Serum ) in the rats was −0.44 ± 0.07‰ at week 2 and −0.36 ± 0.06‰ and −0.36 ± 0.08‰ with a standard-and low-Ca-diet, respectively. This is in the range of findings recently demonstrated in vitro [22] and in various mammalians, including humans [2,9,19]. In addition, we now demonstrate that δ 44/42 Ca is similar in weight-bearing and non-weight-bearing bones, independent of the age and the gender of the animals and the dietary Ca supply.…”

“…δ 44/42 Ca Serum positively correlates with serum biomarkers of bone formation and negatively correlates with bone resorption markers and cortical bone mineral density [21]. In vitro, we recently demonstrated preferential deposition of the light Ca isotope with extracellular matrix calcification by osteoblasts, similar as in other biomineralization processes, and preferential transport of 42 Ca across a proximal tubular epithelial cell barrier, whereas the fractionation process across endothelial cells and enterocytes cells was low [22]. Taken together, the reported findings are highly promising with regard to the application of δ 44/42 Ca Serum and δ 44/42 Ca Urine as a biomarker of bone Ca balance.…”

“…The paracellular pathway depends on the permeability of the tight junctions and the electrochemical gradient between the two sides of the barrier, which is the driving force for passive, energy independent diffusion [ 27 ]. Thus, the light 42 Ca isotope should more rapidly cross the epithelial barrier following the physiological laws of diffusion provided by the Stokes-Einstein equation and Fick’s first law of diffusion [ 22 ]. In animals on a low-Ca-diet, the subtotal Ca uptake may be achieved by increasing the energy dependent transcellular uptake, with less or even no 42 Ca isotope selectivity [ 35 ].…”

“…Bone mineralization, i.e., extracellular matrix calcification, is associated with δ 44/42 Ca isotope fractionation, i.e., with preferential kinetic deposition of the lighter 42 Ca isotope, similar to other biomineralization processes [ 22 ]. The δ 44/42 Ca gradient between bone and serum (δ 44/42 Ca Bone —δ 44/42 Ca Serum ) in the rats was −0.44 ± 0.07‰ at week 2 and −0.36 ± 0.06‰ and −0.36 ± 0.08‰ with a standard- and low-Ca-diet, respectively.…”

DataSheet1.PDF

“…However, osteoclasts mediate the remodeling process via bone matrix resorption (Guo et al, 2021;Jia et al, 2021). However, the balance between bone formation and bone resorption in osteoporotic patients is disrupted, thus resulting in poor osseointegration after prosthesis implantation (Toepfer et al, 2021). So far, various conventional treatments have been attempted to increase the osseointegration of osteoporosis patients after joint replacement, but these treatments have not alleviated the problem (Monotti et al, 2020;Quinzi et al, 2020).…”