Intact Parathyroid Hormone Levels in Renal Insufficiency

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1 Calcif Tissue Int (15) 57:32%335 Calcified Tissue h~emational 15 Springer-Verlag New York Inc. Intact Parathyroid Hormone Levels in Renal Insufficiency V. T. Fajtova, 1 M. H. Sayegh, z N. Hickey, 2 P. Aliabadi, 3 J. M. Lazarus, z M. S. LeBoff 1 1Department of Medicine, ndocrinology Hypertension Division, Brigham and Women's Hospital, Boston, Massachusetts Department of Medicine, Renal Division, Brigham and Women's Hospital, Boston, Massachusetts Department of Radiology, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, Massachusetts Received: 2 February 15 / Accepted: 26 April 15 Abstract. To define the onset of the rise in intact parathyroid hormone (PTH) levels in renal insufficiency, we conducted a cross-sectional study of parameters of mineral metabolism in patients with varying degrees of renal impairment. Using an immunoradiometric assay to measure intact PTH levels, we found elevations in intact PTH levels as creatinine clearance approaches 60 ml/minute (serum creatinine near 1.8) and a significant inverse relationship between indices of renal function and intact PTH levels (r = -0.60, P < for intact PTH and creatinine clearance). Calcium and phosphate levels correlate less strongly with the degree of hyperparathyroidism (r = "0.3, P < for total calcium; r = 0.31, P < 0.05 for phosphate). As a group, only patients with severe renal failure (creatinine clearance <20 ml/minute) had 1,25-dihydroxyvitamin D levels below normal ( [SM] pg/dl, normal range 15-60). Intact and n-terminal PTH measurements correlate well in this patient population with varying degrees of renal insufficiency (r = 0., P < 0.001). Intact PTH can be elevated in patients with mild to moderate renal insufficiency, thus efforts to prevent the development of secondary hyperparathyroidism in renal failure should be undertaken early in the course of renal insufficiency. Key words: Intact PTH -- Renal insufficiency -- Secondary hyperparathyroidism. Varying degrees of renal insufficiency affect over 10 million Americans, and is associated with the development of secondary hyperparathyroidism and subsequently, osteodystrophy. levated parathyroid hormone (PTH) levels in conjunction with other metabolic perturbations of mineral homeostasis contribute to the development of renal osteodystrophy. Despite the increasing recognition of secondary hyperparathyroidism associated with renal insufficiency, the primary abnormalities in calcium metabolism leading to the increased functional demand for PTH secretion are not fully understood. Hypocalcemia, phosphate retention, reduced renal l a-hydroxylation of vitamin D, and skeletal resistance to the actions of PTH are all thought to contribute to the development of secondary hyperparathyroidism in renal insufficiency. With the advent of an assay for the intact PTH (intpth) it has become possible to measure the PTH 1-84 and evaluate parathyroid function with increased accuracy [1]. Intact PTH (1-84) is rapidly cleared from the circulation by peripheral metabolism. Both kidney and liver contribute to the Correspondence to: V. T. Fajtova cleavage of the intact hormone to the N- and C-terminal fragments, with the liver providing two-thirds of this degradation. The liver, however, metabolizes only the intact hormone, and the fragments are predominantly cleared by the kidney, with bone also contributing to the clearance of the biologically active N-terminal fragments. All fragments are filtered at the glomerulus, and the biologically active moieties (intact hormone and N-terminal fragments) are also cleared through peritubular uptake [2-5]. The presence of renal failure and uremia causes a further 25% reduction in hepatic uptake and cleavage of intpth [5]. Thus, renal dysfunction may have some direct effect on the levels of intpth, by decreasing the clearance of this biologically active hormone, and the PTH fragments, which are entirely dependent on renal clearance, may be affected even more. Therefore, older assays, which measure PTH fragments, may be misleading in patients with renal insufficiency. There are discrepancies in the PTH bioactivity and the N- and C-terminal fragment levels [6] in patients with renal failure, and even in the presence of normal renal function, there is discordance between the fall in bioactive and immunoreactive PTH levels after parathyroidectomy in patients with primary hyperparathyroidism [7]. Thus, measurement of intpth levels in patients with renal failure may better reflect the parathyroid gland secretory activity [8] rather than the effect of decreased renal function on the disappearance of PTH fragments from the circulation. However, little is known about the effects of diminished renal function on circulating levels of intpth and the biological impact of PTH excess on bone. Available data indicate that intpth levels are elevated in patients with endstage renal failure [, 10]. N-terminal PTH levels (npth) rise early in renal insufficiency [11] possibly due to decreased renal clearance or increased hormone production. Less is known about intpth levels in early renal insufficiency. There is only one published report examining this issue [12], using and assay for intpth developed by Blind et al. [13]. In this report, Reichel et al. documented a rise in intpth in a group of non-nephrotic patients with mild renal insufficiency, with glomerular filtration rate ranging from 60 to 0 ml/minute when compared with normal controis [12]. We used an assay for the intact hormone, PTH 1-84 (intpth; Allegro TM, Nichols). This is an immunoradiometric assay that is not affected by high concentrations of circulating PTH fragments [14]. This assay has been effective in defining the dynamics of parathyroid secretion in response to changes in serum calcium levels [15, 16], and has been widely used for the clinical and experimental assessment of parathyroid function. Because of the availability of this assay for intpth, its reliability, and its widespread use, we used the assay to define the onset of the rise in intpth levels in patients with renal insufficiency. We conducted a

2 330 cross-sectional study of parameters of mineral metabolism in patients with varying degrees of renal impairment. We found a significant inverse relationship between indices of renal function and intpth levels, with some patients with mild renal insufficiency having elevations in intpth levels. In addition, we made a direct comparison of Intact and N-terminal PTH levels in this group of patients with varying degrees of renal insufficiency, and found an excellent correlation between these two measurements. Materials and Methods xperimental Subjects ~.'-" V. T. Fajtova et al.: Intact PTH in Renal Insufficiency... Int PTH = 65 Nondialyzed patients with renal impairment were recruited from outpatient Renal and ndocrine clinics at the Brigham and Women's Hospital. A total of 66 patients were screened, and 7 were excluded because they were taking medications (vitamin D or estrogens) that may affect the metabolic parameters studied. The final study population consisted of 5 patients, including 22 men and 37 women, from 20 to 81 years of age. The etiology of renal disease in these patients varied, hypertension being most common (n = 34), followed by diabetes mellitus (n = 13), and some of these patients had both diseases (n = 11). ther causes of renal insufficiency included IgA nephropathy (2), Alport's disease (2), nephrolithiasis (2), pyelonephritis (2), and miscellaneous (14). Concomitant diseases included atherosclerotic heart disease () and cerebrovascular disease (3), arthritis (4), hypothyroidism (2), and other unrelated medical conditions (8). ne patient with mild renal insufficiency had proteinuria (286 mg/24 hours) and normal serum albumin, and two with severe renal insufficiency had low serum albumin (3.1 and 2.4 g/dl; normal ). No patients had other laboratory evidence of liver dysfunction. Fourteen patients were taking calcium carbonate as their primary phosphate binder (standard care at our institution), and two were on aluminum containing phosphate binders. ther medications included calcium channel blockers (n = nifedipine, 7 diltiazem, 1 verapamil), furosemide (8), p-blockers (10), AC inhibitors (8), nitrates (6), nonsteroidal antiinflamatory medications (7), cytoxan (3), prednisone (1), bromocriptine (1), and miscellaneous others. Two hypothyroid patients were taking levothytroxine and were biochemically euthyroid. Patients were asked to participate in the study on their routine visits to our offices. Informed consent was obtained. Patients consumed their usual diet prior to study. Blood was collected between :00 a.m. and 12:00 noon. f the 58 study subjects, 3 collected urine for 24-hour creatinine clearance within 60 days of blood sampling. To assess the presence of renal osteodystrophy, 2 subjects had radiographs of clavicles, hands, spine, and pelvis. This protocol was approved by the Committee For The Protection f Human Subjects From Research Risks at Brigham and Women's Hospital. After collection, blood was refrigerated, and processed within 3 hours of collection. Serum calcium concentration was measured immediately using a NVA-7 calcium analyzer, which measures total and ionized calcium levels using calcium-selective electrodes (Nova, Waltham, MA). The ionized calcium levels were normalized to a serum ph of 7.4. Intact PTH concentrations were measured; (normal range pg/ml) using the Allegro TM immunoradiometric assay (Nichols Institute, San Juan Capistrano, CA) in our clinical research laboratory, npth concentrations were measured in our research laboratory using an intact N-terminal specific investigational radioimmunoassay (RIA) (Nichols Institute; normal range 8-24 pg/mi). Serum 25 hydroxyvitamin D (25vitD) and 1,25vitD levels were measured with competitive protein assays (Nichols Institute). Serum and urine creatinine and serum phosphate levels, alkaline phosphatase activity, and other routine chemistries were also obtained. Creatinine clearance was calculated from these data. Radiographs were evaluated by a member of the radiology department at Brigham and Women's Hospital specializing in bone disorders (PA), who was blinded to the patient's renal function. Parathyroid bone disease was classified as none (0), mild (1), or severe (2). Radiographs that demonstrated subperiosteal resorp Creatinine Clearance (ml/min) Fig. l. Relationship between intpth and creatinine clearance. By univariate regression r = -0.53, P < 0.001, n = 3. IntPTH was not noted to rise out of the normal range until the creatinine clearance fell to less than 60 ml/minute. tions, and/or osteosclerosis, were classified as indicating mild parathyroid bone disease; those that showed frank erosions and/or lesions consistent with brown tumor were classified as having severe PTH related skeletal changes. nly data that included 24-hour creatinine clearance were used for analysis involving renal function. Data for all patients who had both intact and n-terminal PTH levels determined were used for comparison of these values. Patients were divided into three groups according to the severity of their renal impairment: patients with creatinine clearances greater than 60 ml/minutes were considered to have mild renal insufficiency, those with creatinine clearances between 20 and 60 ml/minutes were considered to have moderate renal impairment, and patients with creatinine clearances less than 20 were considered to have severe renal failure. The Kruskal-Wallace test for populations with nonparametric distributions was used to determine significance of differences among groups. Correlation analysis was used to evaluate a relationship between pairs of variables, and multiple regression was used to determine the influence of several variables on intpth and alkaline phosphatase. The curve in Figure 1 was generated by logarithmic regression of the data. Data analysis was assisted by the CLINF data management and analysis system in conjunction with SAS statistical package [17]. Data are expressed as mean -+ standard error of the mean (m -+ SM). Results Dependence of intpth and ther Parameters of Bone Metabolism on Renal Function The relationship between intpth and selected parameters of mineral metabolism are shown in Table 1. The single most important determinant of intpth levels was renal function (Fig. 1). By simple regression analysis, intpth correlated equally well with both creatinine clearance and serum cre-

3 V. T. Fajtova et al.: Intact PTH in Renal Insufficiency 331 Table l. Correlation relationships of bone-related metabolic parameters a r r 2 P n intpth and creatinine clearance < intpth and serum creatinine < intpth and total calcium intpth and ionized calcium intpth and phosphate intpth and 1,25vitD intpth and aluminum Total calcium and creatinine clearance Phosphate and creatinine clearance < ,25vitD and creatinine clearance Alk-phos and intpth < Alk-pbos and creatinine clearance Correlation analysis was performed on all subjects who were enrolled in this study, including some who did not have all data atinine levels (r = 0.60, P < for creatinine clearance: r = 0.52, P < for serum creatinine). The correlation between intpth and the Log (creatinine clearance) was slightly stronger than between intpth and creatinine clearance (r = -0.61, P < 0.001). As shown in Figure 1, intpth levels rose out of the normal range (10-65 pg/ml) when the creatinine clearance fell to 60 ml/minute. Serum calcium showed a weaker, but still significant correlation with intpth levels (r = -0.3, P < 0.01), but the relationship between intpth levels and phosphate, 1,25vitD and aluminum levels was not statistically significant (Table 1). Total and ionized serum calcium concentrations both fell significantly with declining renal function, and serum phosphate concentration rose. In our study group there were no significant correlations between intpth levels and age or weight (data not shown). When analyzed by multiple regression analyses, creatinine clearance was the only independent determinant of the variation in intpth levels, whereas calcium, phosphate, and 1,25vitD did not contribute independently to the elevation of intpth levels with diminishing renal function (Table 2). Thus, the strongest determinant of intpth levels was renal function. Analysis of Parameters of Bone Metabolism by Groups of Severity of Renal Failure The data comparing intpth levels, as well as calcium, phosphate, 1,25vitD, and alkaline phosphatase concentrations among the three different groups of subjects with worsening renal function are summarized in Table 3. All of these parameters changed significantly as renal function worsened. Levels of intpth (Fig. 2), alkaline phosphatase activity, and serum phosphate (Fig. 3) were significantly elevated in the group of patients with severe renal failure as compared with the two other groups (P < 0.01), whereas the 1,25vitD levels were significantly lower in the group with severe renal failure than in patients with mild or moderate renal insufficiency (Fig. 3). ffects of Renal Insufficiency on Bone Serum alkaline phosphatase activity, as an index of bone turnover, was very tightly correlated with intpth levels (r = 0.77, P < 0.001), and less tightly correlated with creatinine clearance (r = -0.43, P < 0.01). n multiple regression analyses, the concentration of intpth was the only variable contributing independently to changes in alkaline phosphatase (Tables 1 and 2). nly a few patients in our study group had radiographic findings of hyperparathyroidism. No patients with mild renal insufficiency had positive radiographs, and 2 out of the 22 with moderate and severe renal failure who were studied radiographically had mild bone changes consistent with hyperparathyroidism. ffect of Diabetes Mellitus and Calcium Channel Blockers on intpth Levels Since patients with diabetes mellitus have been reported to have suppressed intpth levels [18, 1], we compared intpth levels of patients with diabetes mellitus to those with comparable degree of renal insufficiency. There was no significant difference in the intpth levels between diabetic and nondiabetic patients in our patients (data not shown). Likewise, patients who were on calcium-channel blockers did not have intpth levels that differed from those who were taking other antihypertensive medications and those who were not hypertensive (data not shown). Thus, these patients were included in the general study group. ur study populations may not be large enough to show a significant difference between these groups. Correlations of intpth with npth Levels Concentrations of intpth and npth are highly correlated regardless of the degree of renal insufficiency, even in the Table 2. Multiple regression relationships of bone-related metabolic parameters a Variable r 2 p intpth Creatinine clearance Total calcium Serum phosphate ,25(H) 2 vit D verall Alkaline phosphatase intpth Creatinine clearance Total calcium verall a nly subjects with available creatinine clearance values were included in this analysis

4 332 V. T. Fajtova et al.: Intact PTH in Renal Insufficiency Table 3. Comparison of metabolic parameters relating to calcium homeostasis by severity of renal insufficiency a Group Mild Moderate Severe analysis b (n = 13) (n = 14) (n = 18) Creatinine clearance (ml/min) (61-120) (20-57) (3-18) intpth (pg/ml) Sev > mod = mild (normal 10-65) (18-7) (17-383) (57-627) P < 0.01 Calcium (mg/dl).3 _ Sev < mild (normal ) ( ) ( ) (7.1-.4) P < 0.01 Phosphate (mg/dl) Sev > mod = mild (normal ) (2.-4.5) ( ) ( ) P < ,25(H)2 vit D 3 (pg/ml) Sev < mod = mild (normal 15-60) (5-80) (15-66) (0-22) P < 0.01 Alk-phos (IU/liter) Sev > rood = mild (normal ) ( ) (66-126) (63-348) P < 0.01 a Numbers represent mean -+ SM; numbers in parentheses represent range for each group b The Kruskal-Wallace test for nonparametric distributions was used to determine differences between groups This analysis was performed on samples from patients who were screened for this study, and some of these patients were excluded from other components of the study because they were taking medications that could interfere with mineral metabolism. 500 Discussion I 0 ~6 t- 400,_ MILD MDRAT SVR Fig. 2. IntPTH levels divided according to severity of renal insufficiency. IntPTH levels were significantly elevated in the group with severe renal failure as compared with the other two groups (P < 0.05). Mild refers to the group of patients with creatinine clearances >60 ml/minute, patients with creatinine clearances between 20 and 60 ml/minute were considered to have moderate impairment in renal function, and patients with creatinine clearances <20 ml/minute comprise the group with severe renal failure. high range (n = 37, r = 0.0) (Fig. 4). The ratio of intpth to npth to npth was , and the relationship between the two PTH assays can best be defined by the following equation: npth = x intpth, Though the existence of secondary hyperparathyroidism in renal failure is well established, the onset and initial defect in mineral metabolism associated with renal impairment are less well characterized. Previous studies have shown that the active N-terminal and inactive C-terminal, or midmolecule PTH fragments, are elevated in mild renal insufficiency, near creatinine clearances of 60 ml/minute [1, 20]. We have demonstrated that this holds true not only for PTH as measured by RIA, which may be effected by circulating PTH fragments, but also for intpth (intact PTH 1 to 84) as measured by the immunoradiometric assay [1]. As intpth is the molecule predominantly secreted by the parathyroid gland, and its clearance is least affected by renal insufficiency, our data suggest the presence of parathyroid hypersecretion early in renal failure, rather than merely impaired clearance of PTH and its fragments. This elevation in intpth levels reflects a defect in mineral metabolism which occurs early in renal insufficiency. Hypocalcemia, as evaluated by currently available static techniques for measuring total and ionized calcium concentrations, does not occur until after the PTH levels are elevated [10]. Impaired clearance of phosphate resulting in impaired la-hydroxylation and decreased gastrointestinal calcium absorbtion has been implicated in secondary hyperparathyroidism of renal insufficiency [20--23]. 1,25vitD facilitates calcium absorbtion and directly suppresses parathyroid gland secretion [, 10, 23-26]. Histologic studies have demonstrated a decreased responsiveness of bone to the resorptive action of PTH in uremic patients [28]. Thus, 1,25vitD may represent a link between early renal dysfunc-

5 V. T. Fajtova et al.: Intact PTH in Renal Insufficiency C "ID ~n v "5 k_ 1 0'3-6 ~3 F- 10 4I. 6 0 v r- 6 > 40 X 0 k- " r :5 20 it') cn "T "I " T w I T m 1 MILD MDRAT SVR MILD MDRAT SVR b. d. 35o m no 7 % 3ooy I / / 1 v 1 r- c- (3_ -n k_ ID (/) m MILD MDRAT SVR Fig, 3. Comparison of total serum calcium (a), phosphate (b), 1,25vitD (c) and alkaline phosphatase (d) concentrations. Groups with mild. moderate, and severe renal insufficiency are as described for Figure 2. All of these parameters changed significantly, as renal function worsened. Total serum calcium concentration was significantly lower in the group with severe renal faiture than those in mild =o n 0J r- "- I00 t 6 o 50 MILD MDRAT SVR renal insufficiency (P < 0.0l). The serum phosphate concentration and alkaline phosphatase activity were significantly elevated in the group with severe renal failure whereas the 1,25vitD levels were lower compared with the other two groups with less severe renal dysfunction (P < 0.01). tion and the resulting hyperparathyroidism. arly phosphate retention may lead to inadequate activation and action of vitamin D, which may contribute to end organ resistance to PTH action and hypocalcemia. In addition, reduced sensitivity of the parathyroid glands to the suppressive effects of calcium may lead to chronic stimulation and increase in parathyroid cell mass. Chronically, this leads to parathyroid overstimulation, hyperplasia, and hypersecretion. We demonstrated a significant correlation between creatinine clearance and serum calcium levels as well as creat-

6 334.. I F r._ L- Z I ID 0 I I I I I II Intact PTH (pg/ml) Fig. 4. Correlation of intpth with npth. IntPTH and npth are highly correlated regardless of the degree of renal insufficiency (r = 0.0), even in the high range. This analysis includes samples from all patients who were screened for this study (n = 37). Some of these patients were excluded from the rest of the study because they were taking medications that may interfere with mineral metabolism. inine clearance and phosphate levels, however, these levels were only significantly abnormal in the group of patients with severe renal insufficiency (creatinine clearance less than 20 ml/minute). At this degree of renal failure, parathyroid hyperfunction is well established. Similarly, whereas there is a significant correlation between 1,25vitD levels and creatinine clearance when the whole study population is analyzed (P < 0.05), 1,25vitD levels are low only in the group of patients with severe renal insufficiency (Tables 1, 3). Functional hyperparathyroidism may compensate in mild and moderate renal insufficiency, and drive lc~-hydroxylation of vitamin D, without notable changes in serum total and ionized calcium. Multivariate analysis of several parameters of mineral metabolism identifies creatinine clearance as the strongest independent predictor of elevated intpth levels, with serum calcium playing a lesser role, and phosphate and 1,25vitD having little independent contribution (Table 2). However, in more advanced renal insufficiency, calcium, phosphate, and 1,25vitD levels are all abnormal, and thus may contribute independently to the elevations in intpth levels. Therefore, other than in declining renal function itself, we have not been able to identify any static metabolic parameters that may initiate the rise in serum intpth levels in patients with mild renal insufficiency. Alkaline phosphatase activity, as a marker of bone turnover, correlates well with the degree of hyperparathyroidism, being most elevated in the group of patients with severe renal insufficiency. Uremic patients demonstrate skeletal resistance to the action of PTH [28], however, the elevations of alkaline phosphatase in our subjects suggest that there may be increased resorption of calcium from bone V. T. Fajtova et al.: Intact PTH in Renal Insufficiency in this group of nondialized patients. By multivariate analysis, intpth rather than creatinine clearance appears to be a major independent determinant of alkaline phosphatase activity (Table 2), suggesting that the skeletal effects of renal failure are largely PTH mediated. Bone resistance to PTH action in uremic patients may serve to protect the bone by attenuating bone resorption, but may also exacerbate the hyperparathyroidism by diminishing the calcemic response to elevations in PTH levels. Uremic and hyperplastic parathyroid gland tissue may secrete increased proportion of PTH fragments [2], thus elevating the npth out of proportion to the intpth. In addition, the clearance of intpth may be influenced by the renal dysfunction to a lesser degree than npth [2]. However, we found an excellent correlation between the intpth levels and PTH as measured by a radioimmunoassay specific for the N-terminal portion of the PTH molecule, suggesting that the N-terminal measurement is valid in the whole spectrum of renal failure. IntPTH appears to accurately assess biologically significant PTH levels, and not to be effected by circulating PTH fragments. Thus, studies utilizing the older, N-terminal assay for determination of PTH levels do reflect the true status of the calcium homeostatic system in patients with renal failure. ur data may underestimate the severity of renal impairment in this patient population, since the creatinine clearance overestimates the glomerular filtration rate when compared with glomerular filtration rate measured by inulin clearance [30]. This holds true for all degrees of renal impairment. Therefore, static measurements demonstrate abnormal calcium, phosphate, and 1,25vitD levels even later in renal insufficiency than is estimated here (as well as in most of the studies performed to investigate this question). In this study, the creatinine clearance was not corrected for body habitus. Such a correction may redistribute the data, but should not alter the overall findings. We included serum creatinine levels in our analysis, to allow comparison with easily accessible clinical measurements. We found intact PTH levels to be elevated in nonnephrotic patients with mild to moderate renal failure, before abnormalities in serum calcium, phosphate, or vitamin D become apparent. Since intpth is less dependent on renal function for its clearance than both active and inactive PTH fragments, these observations represent parathyroid hypersecretion in patients with mild to moderate renal insufficiency. Dynamic studies that perturb calcium, phosphate, 1,25vitD, and PTH levels in a way that mimic the everyday life of our patients, would be necessary to definitively elucidate the pathogenesis of hyperparathyroidism in early renal failure. These studies emphasize the necessity to address the development of secondary hyperparathyroidism early in the course of renal insufficiency, both clinically and experimentally. Acknowledgments. CLINF at Brigham and Women's Hospital, Boston, MA, is supported by the GCRC Grant 5-M1-PR We would like to express our appreciation to Mr. Ray Gleason for his assistance with the statistical analysis of our data. References 1. Nussbaum SR, Zahradnik RJ, Lavigne JR, Brennan GL, Nosawa-Ung K, Kim LY, Keufmann HT, Wang CA, Potts JT, Segre GV (187) Highly sensitive two-site immunoradiometric

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