The hypercalcemia of sarcoidosis has been causally linked to the endogenous overproduction of a vitamin-D-related hormone. An increase in the assayable serum concentration of 1,25-(OH)2-D has even been documented in an anephric patient with sarcoidosis and hypercalcemia suggesting an extrarenal site of synthesis of the hormone. Our preliminary data suggest that the macrophage, a major constituent of the ubiquitous noncaseating granulomata of sarcoidosis, is the synthentic source of a biologically-active vitamin D sterol. Incubation of primary cultures of pulmonary alveolar macrophages (PAMs) from a patient with active sarcoidosis with (3H)25-OH-D3 on two separate occasions resulted in production of more polar substance. The labelled metabolite co-chromatographed with authetic 1,25-(OH)2-D3 on straight-phase HPLC in three different solvent systems and demonstrated a binding affinity for the specific 1,25-(OH)2-D3 receptor comparable to that of (3H)1,25-(OH)2-D3. In light of these findings the major objectives of the proposed work are threefold: 1) confirm our initial observation that PAM production of 1,25-(OH)2-D3, or a closely related compound, is specific for sarcoidosis; 2) identify the molecular structure of the PAM-derived metabolite my means including mass spectral analysis; and 3) correlate synthesis of the metabolite by PAMs in vitro to disease-related clinical and biochemical abnormalities demonstrated by the host in vivo. In addition, the research is designed to determine the kinetics and substrate specificity of the PAM enzyme in vitro and to explore the feasability of studying 25-OH-D3 metabolism in a more accessible cell and precursor to the tissue macrophage, the circulating monocyte. We believe this work will advance our understanding of cellular mechanisms responsible for the vitamin D sterol production in this disease.
