A new method for in vivo analysis of parathyroid hormone-calcium set point in mice

Although methods for measuring the parathyroid hormone (PTH)-calcium set point in vivo in humans and large animals exist, translating such methods to the increasingly important mouse model poses considerable challenges. We also found that manipulation of dietary calcium does not yield sufficiently high or low serum calcium levels to achieve the minimum and maximum PTH levels needed for set point estimations. Therefore, we developed a new method for in vivo evaluation of the relative set point in mice. Intraperitoneal injection of calcium gluconate caused progressive increases in serum calcium over 120 minutes, with corresponding decreases in serum PTH levels. Intraperitoneal injection of Na2-EGTA caused a nadir of serum calcium at 30 minutes and a corresponding peak value of serum PTH. The lowest and highest serum calcium concentrations achieved were 6.7 mg/dl and 10.0 mg/dl, respectively. Linear regression analyses indicated high correlation coefficients (serum calcium vs. serum PTH; r = 0.969). To obtain the additional data points needed for set point estimation, blood was collected 30 minutes after intraperitoneal injection by tail nicking from three mice once a day, on 8 consecutive days, using multiple doses of calcium gluconate or Na2-EGTA. The lowest and highest serum calcium concentrations achieved were 5.0 mg/dl and 11.4 mg/dl. Maximum and minimum PTH levels were indeed observed, and a sigmoidal curve with a set point of 7.8 mg/dl was readily generated by the four-parameter model that was fit using a nonlinear mixed effects statistical approach. This protocol for in vivo set point analysis should be applicable in the future study of multiple genetically engineered and pharmacologically treated mouse models.