increase in PTH secretion, and that the maintenance of a normal serum calcium concentration is in part related to bone and kidney resistance to the biological actions of PTH. In particular, the net bone calcium release, as assessed by the fasting urine calcium and urine creatinine ratio (UCa/UCr), was lower in the normocalcemic than in the hypercalcemic subgroup. The markers of bone turnover were also lower in the normocalcemic than in the hypercalcemic subgroup. The ability of the renal tubule to reabsorb calcium was lower in the former than the latter group of patients. In addition, the ability of PTH to decrease tubular phosphate reabsorption and stimulate synthesis of 1,25-dihydroxyvitamin D is also blunted in patients who remain normocalcemic, compared with those who are hypercalcemic. Therefore, at least 3 PTH-dependent functions of the kidney are attenuated in NPHPT patients despite an identical primary hypersecretion of PTH, demonstrating a partial renal resistance to the physiological actions of PTH. However, the nephrogenous cAMP secretion is indistinguishable between normocalcemic and hypercalcemic matched subgroups, indicating that the amount of serum bioactive PTH (1–84) is the same in both subsets of patients. Estrogen supply appears to induce some degree of bone and kidney resistance to the effects of PTH in PHPT women, similar to what is observed in patients with NPHPT. However, patients with NPHPT had a higher BMI than hypercalcemic patients. This higher BMI could explain a higher residual estrogen production related to a greater mass of adipose tissue, which is an important site of estrogen biosynthesis in postmenopausal women [21].
In line with the potential effect of estrogens as inductors of PTH resistance, an orphan adhesion G protein-coupled receptor, GPR64/ADGRG2, has been discovered expressed in human normal parathyroid glands and overexpressed in parathyroid tumors from PHPT patients. Interestingly, GPR64 physically interacts with CASR and its activation increases PTH release from human parathyroid cells at a range of calcium concentrations [22].
4. Polymorphisms of CASR and PTH genes in NPHPT: A986S polymorphism of the CASR has been evaluated as a determinant of PTH resistance in NPHPT and asymptomatic PHPT [23]. The CASR A986S polymorphism has been extensively linked to PTH resistance and higher calcium levels in population-based studies. Though A986S CASR variants do not seem to be major genetic determinants for the development of PHPT, even in earlier or asymptomatic forms, in NPHPT, only the A986S genotype was an independent predictor of PTH levels even after adjusting for major confounding factors such as vitamin D levels and serum calcium concentrations. Therefore, PTH levels in NPHPT may be partially regulated by the A986S polymorphism, acting as a resistance factor due to a relative loss of CASR function.
PTH gene polymorphisms have been associated with a disease severity in classical PHPT. In asymptomatic PHPT patients, the GG genotype of the rs6254GA polymorphism was associated with a significantly higher PTH level and lower bone mineral density (BMD) at the femoral neck, proximal femur, and lumbar spine. The association failed to be demonstrated in NPHPT patients [24].
Diagnosis
The diagnostic approach to an “isolated” elevated PTH (Fig. 1) may be a step-by-step process: as the first step, causes of evident SHPT should be ruled out; the second step considers the diagnosis of NPHPT if no causes of SHPT are identified and if serum calcium is in the upper half of the normal range; at this point, in order to complete the diagnostic workup, PHPT-related bone and kidney diseases should be evaluated. Some conditions are evident in which it is possible to elevate PTH levels, while others are asymptomatic and need to be investigated. Among these conditions, vitamin D insufficiency occurs frequently.
This diagnostic workup to exclude causes of SHPT (Table 1) has been strongly recommended in the 2 most recent guidelines on the diagnosis and management of asymptomatic PHPT published in 2009 [1] and 2014 [25]. First-line exploration may include serum calcium, albumin, phosphate, PTH, and 25(OH)D, 24-h calciuria, serum creatinine, and estimated glomerular filtration rate (eGFR). A second-line exploration will include ionized calcium, 24-h phosphaturia and creatininuria (and calculation of TmP/GFR), serum alkaline phosphatase, TSH, and magnesium.
In performing the biochemical screening, a number of items should be considered:
•Serum calcium, phosphate, and PTH undergo marked circadian variations. Evaluation should always be performed in the morning after an overnight fasting in asymptomatic subjects. In emergency rooms, when symptoms of hyper- or hypocalcemia are evident, evaluation needs to be immediate. Ingestion of food containing significant amounts of calcium will increase serum calcium and decrease PTH; salt, protein, and glucides may increase urine calcium excretion.
•Ionized calcium: when serum total and ionized calcium values in patients with PHPT are compared, ionized calcium values are more frequently elevated than total calcium values [21, 26]. Ionized calcium is highly influenced by the pH; the affinity of albumin for calcium increases when the pH increases, and decreases when the pH decreases. Therefore, ionized calcium will be lower in the case of alkalosis and higher in the case of acidosis. Determination of ionized calcium suffers from frequent preanalytical problems (avoiding venous stasis and tourniquet use, respect for anaerobiosis).
Table 1. Causes of SHPT, which should be ruled out in the diagnostic workout of NPHPT; biochemical markers, normal values, and main treatments are suggested
•Calciuria measured on 24-h urine collections (ideally expressed in mg/kg body weight/24 h) represents intestinal absorption of calcium, while the second morning void obtained after an overnight fast, expressed as a ratio to creatininuria, represents an index of bone resorption as the measured calcium may only come from bone. The reference range of urine calcium excretion has been established in normal subjects whose calcium intake was approximately 1,000 mg/day; therefore, calcium intake should be considered and verified concomitantly with urine collection for 24-h calciuria. Questionnaires are freely available online (for example from the International Osteoporosis Foundation, www.iofbonehealth.org/calcium-calculator).
•Measurement of phosphatemia needs to avoid hemolyzed samples and reference values vary with age: 1.50–2.30 mmol/L in newborns less than 1 month old; 1.50–2.00 mmol/L from 1 month to 2 years; 1.40–1.70 mmol/L from 2 to 12 years; 1.00–1.50 mmol/L from 12 to 16 years, and 0.80–1.40 in adults.
•Phosphaturia, best calculated as TmP/GFR, should be measured in case of hypophosphatemia to determine whether this anomaly is due to renal leak (tubular acidosis, FGF23 excess) or to another cause. A low TmP/GFR in the presence of hypophosphatemia suggests renal phosphate leak.
•Pregnancy: NPHPT should not be diagnosed in pregnant women. Hypoalbuminemia, increased GFR, transplacental transfer of calcium, and increased levels of estrogen all contribute to lowering gestational serum calcium levels and the masking of HPHPT [27].
•Vitamin D replacement: subjects with elevated PTH and normal calcium levels and vitamin D insufficiency may fall into 1 of the following 3 categories: (a) they may have HPHPT and their low calcium level is secondary to the impaired vitamin D status: such patients may become hypercalcemic when their vitamin D is replaced; (b) some of these patients may have elevated PTH due to their impaired vitamin D status: such subjects are expected to achieve