Heinrich Schmidt-Gayk

Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1-84: Implications for improvement of accurate assessment of parathyroid function

We developed a novel immunoradiometric assay (IRMA; whole parathyroid hormone [PTH] IRMA) for PTH, which specifically measures biologically active whole PTH(1–84). The assay is based on a solid phase coated with anti‐PTH(39–84) antibody, a tracer of125I‐labeled antibody with a unique specificity to the first N‐terminal amino acid of PTH(1–84), and calibrators of diluted synthetic PTH(1–84). In contrast to the Nichols intact PTH IRMA, this new assay does not detect PTH(7–84) fragments and only detects one immunoreactive peak in chromatographically fractionated patient samples. The assay was shown to have an analytical sensitivity of 1.0 pg/ml with a linear measurement range up to 2300 pg/ml. With this assay, we further identified that the previously described non‐(1–84)PTH fragments are aminoterminally truncated with similar hydrophobicity as PTH(7–84), and these PTH fragments are present not only in patients with secondary hyperparathyroidism (2°‐HPT) of uremia, but also in patients with primary hyperparathyroidism (1°‐HPT) and normal persons. The plasma normal range of the whole PTH(1–84) was 7–36 pg/ml (mean ± SD: 22.7 ± 7.2 pg/ml, n = 135), whereas over 93.9% (155/165) of patients with 1°‐HPT had whole PTH(1–84) values above the normal cut‐off. The percentage of biologically active whole PTH(1–84) (pB%) in the pool of total immunoreactive “intact” PTH is higher in the normal population (median: 67.3%; SD: 15.8%; n = 56) than in uremic patients (median:53.8%; SD: 15.5%; n = 318; p < 0.001), although the whole PTH(1–84) values from uremic patients displayed a more significant heterogeneous distribution when compared with that of 1°‐HPT patients and normals. Moreover, the pB% displayed a nearly Gaussian distribution pattern from 20% to over 90% in patients with either 1°‐HPT or uremia. The specificity of this newly developed whole PTH(1–84) IRMA is the assurance, for the first time, of being able to measure only the biologically active whole PTH(1–84) without cross‐reaction to the high concentrations of the aminoterminally truncated PTH fragments found in both normal subjects and patients. Because of the significant variations of pB% in patients, it is necessary to use the whole PTH assay to determine biologically active PTH levels clinically and, thus, to avoid overestimating the concentration of the true biologically active hormone. This new assay could provide a more meaningful standardization of future PTH measurements with improved accuracy in the clinical assessment of parathyroid function.

Circannual Rhythms and Interactions of Vitamin D Metabolites, Parathyroid Hormone, and Biochemical Markers of Skeletal Homeostasis: A Prospective Study

Recent studies suggest a circannual pattern of bone turnover. To further investigate the underlying mechanisms, 41 healthy subjects (25‐80 years old) living in a southwestern German city were studied prospectively over a period of 18 months. Participants were examined every 4 weeks, and blood and urine samples were obtained on each visit. The following parameters were measured: serum 25‐hydroxyvitamin D3 [25(OH)D3], 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3], and parathyroid hormone (PTH), as regulators, and serum total alkaline phosphatase (TAP), bone‐specific alkaline phosphatase (BAP), urinary total pyridinoline (PYD), deoxypyridinoline (DPD), and the aminoterminal telopeptide of collagen type I (NTX), as biochemical markers of bone turnover. The presence of significant circannual rhythms for the various markers was tested using the Pharmfit method. In the total group, 25(OH)D3, 1,25(OH)2D3, and PTH as well as BAP, PYD, DPD, and NTX showed a significant seasonal variation. 25(OH)D3 revealed the highest amplitude (38.0%) with an acrophase in August. Levels of the biochemical markers and of PTH were highest in winter with amplitudes of up to 17.7% (DPD). Results were most pronounced in premenopausal women, in subjects <50 years of age, and in subjects who did show a significant individual rhythm in 25(OH)D3 levels. No differences were found regarding other anthropometric or life style factors. Correlation analyses revealed strongest associations between the amplitudes of a vitamin D metabolite and a biochemical marker in premenopausal women. We conclude that specific markers of bone turnover show significant circannual rhythms. These changes are related directly to variations in the hormonal regulation of skeletal homeostasis. In postmenopausal women and in men, other effects may superimpose the circannual variation of biomarkers of bone turnover.

Changes in Bone Turnover Induced by Aerobic and Anaerobic Exercise in Young Males

Physical activity is considered an important factor in attaining bone mass. However, the mechanisms by which exercise affects bone metabolism are not completely understood. The present study was performed to investigate the effects of aerobic and anaerobic exercise on bone turnover. Twenty healthy young males (aged 20–29 years) were followed through an 8‐week program of aerobic (n = 10) and anaerobic training (n = 10). Ten age‐matched individuals served as controls. Serum bone‐specific alkaline phosphatase (BAP), serum osteocalcin (OC), and urinary pyridinoline (Pyd) and deoxypyridinoline (Dpd) were determined as indices of bone metabolism. After 4 weeks of aerobic training, serum BAP and OC (p < 0.01), and urinary Pyd (p < 0.001) and Dpd (p < 0.01) were significantly reduced. After 8 weeks, BAP and OC levels had returned to baseline values, whereas the urinary cross‐link excretion remained low. In the anaerobic training group, elevated levels of BAP (p < 0.05 vs. week 4), OC (p < 0.05 vs. week 4), and Pyd (p < 0.01 vs. week 0) were observed after 8 weeks of exercise. Changes in urinary Pyd and Dpd (week 0 vs. week 8) were positively correlated with changes in the mean power level in the Wingate test, a parameter of the anaerobic performance capacity (r = 0.50 and r = 0.55, p < 0.01, respectively). In the controls, no significant changes in biochemical markers were observed. We conclude that aerobic and anaerobic training excert different effects on bone metabolism. While aerobic training led to changes compatible with reduced bone resorption activity, anaerobic training seems to result in an overall accelerated bone turnover. Therefore, the impact of physical activity on bone turnover may depend on the kind of exercise performed.

Comparison of Intermittent and Continuous Oral Administration of Calcitriol in Dialysis Patients: A Randomized Prospective Trial

Intermittent bolus administration of calcitriol--i.e., 1,25-dihydroxycholecalciferol or 1,25-(OH)2D3--is highly efficacious in dialysis patients. In experimental studies, intermittent administration of calcitriol is superior to continuous administration in suppressing preproparathyroid hormone (PTH) mRNA and circulating PTH concentrations. In a randomized, prospective, open multicenter trial 45 dialysis patients with elevated 1,84-iPTH (> or = 20 pmol/l, normal 1-6 pmol/l) levels were randomly allocated to daily administration of 0.75 microgram calcitriol (continuous) or twice weekly administration (intermittent); the two protocols provided an identical total weekly doses of 5.25 micrograms calcitriol. Patients were dialyzed with a dialysate Ca concentration of 1.75 mmol/l and had oral CaCO3 or Ca acetate. 1,84-iPTH (immunoradiometric assay) and serum Ca and Pi levels were measured weekly. At the beginning of the study, the median 1,84-iPTH value was 37 pmol/l (range 20-115) in the intermittent versus 36 pmol/l (range 21-72) in the continuous calcitriol group. After 2 weeks, the median 1,84-iPTH level was 18.5 pmol/l (range 1.4-106) versus 18 pmol/l (range 1.2-48). After 12 weeks, 11 of 21 of the patients in the intermittent and 18 of 24 patients in the continuous group had reached the treatment goal, i.e., 1,84-iPTH < or = 10 pmol/l without hypercalcemia or hyperphosphatemia. There were seven episodes of hypercalcemia (> 2.7 mmol/l) in the intermittent versus two in the continuous group; the mean peak Ca level was 2.8 mmol/l (range 2.76-3.0) versus 2.9 mmol/l (range 2.74-3.06). There were 21 versus 17 episodes, respectively, of hyperphosphatemia (> 2.2 mmol/l).

Measurement of vitamin D and its metabolites (calcidiol and calcitriol) and their clinical significance

The serum concentration of vitamin D will only give information about the recent exposure to either nutritional vitamin D or to recent vitamin D production in the skin. Within hours vitamin D is removed from the circulation and reappears again a few hours later as 25(OH)D. Measurements of vitamin D therefore are not useful to judge the vitamin D status of man. Plasma concentrations of 25-hydroxyvitamin D (25(OH)D) are the best markers of imminent or existing vitamin D deficiency. Suboptimal vitamin D supply (or 25(OH)D plasma concentrations) are observed in most European countries and North America from November to April. Vitamin D substitution is recommended to persons with a serum concentration of 25(OH)D below 50 nmol/L (20 micrograms/L). Plasma concentrations of 1,25(OH)2D3 (calcitriol) depend mainly on renal function, concentrations of intact PTH and the supply of the organism with calcium and phosphate. High PTH, low calcium and low phosphate supply are the main stimulators of the production of calcitriol. Vitamin D substitution, or if necessary, therapy with active vitamin D metabolites or analogs, e.g. calcitriol or alfacalcidol, are often necessary in hypercalcemic persons with low serum concentrations of calcitriol, e.g. in patients with renal failure. Disorders with low or high vitamin D metabolite concentrations in serum are described in this paper.

Performance evaluation of automated assays for β-CrossLaps, N-MID-Osteocalcin and intact parathyroidhormone (BIOROSE Multicenter Study)

Abstract Introduction: Biochemical markers of bone metabolism have been mainly determined manually until now and the precision and accuracy of these methods have not always been satisfactory. This has been shown in several external quality assessment schemes (EQAS). Objective and study design: A study named BIOROSE was undertaken to evaluate new automated assays for serum markers of bone metabolism. The main focus was to evaluate the assay performance in a multicenter setting with 20 laboratories participating in Germany. The evaluation consists of a familiarization phase to determine precision and accuracy and an EQAS to evaluate the comparability between laboratories. Materials: The parameters β-CrossLaps (CTX), N-MID-Osteocalcin (OC) and intact parathyroid hormone (PTH) were measured with reagents including calibrators and control sera obtained from Roche Diagnostics, Mannheim, Germany, with electrochemiluminescence immunoassays (ECLIA) on the automated analyzer Elecsys 2010. Results: We calculated for the control samples, PCB 1-3, the mean and median values from the measured values of all participating laboratories and used these as target values. From these target values, a recovery range for the participating laboratories was calculated for β-CrossLaps, OC and intact PTH of better than 80–126% for PCB 2 and PCB 3, and for PCB 1 (low concentration range) for β-CrossLaps 79–129%, OC 90–120% and intact PTH 78–126%. The between-day imprecision was 2.4–7.2% for β-CrossLaps, 1.1–5.9% for OC and 1.7–5.5% for intact PTH in the elevated range (sample PCB 2). In the EQAS, the inter-laboratory imprecision for β-CrossLaps in the sample with a value of 0.8 ng/ml (above the upper limit of normal, which is 0.6 ng/ml) was 9.8% on day 1 and 9.7% on day 2. Conclusion: The performance evaluation of automated assays for β-CrossLaps, N-MID-Osteocalcin and intact parathyroid hormone in the BIOROSE multicenter study showed that the participating laboratories had no problems in setting up these methods and they yielded results for precision and accuracy that are superior to results achieved in external quality assessment schemes for manually performed methods. In addition, at the clinically important decision level of the upper limit of the normal range, all three tested analytes gave precise results that improved medical decisions.

Skeletal Changes and Growth in Experimental Uremia

Longitudinal growth; bone and growth zone histology; growth cartilage and bone mineralization (tetracycline technique); bone Ca content (neutron activation analysis); bone radiology; serum and urine chemistry; urinary cAMP and serum 25-OH-vitamin D3 were studied in a long-term model of experimental uremia in the rat. Uremia was induced by two-stage subtotal nephrectomy with irradiation of the remaining parenchyma. Ccr in the experimental group was 113 +/- 5.8 micron1/min X 100 g (19.8% of controls) and serum creatinine 1.67 +/- 0.04 mg% (5.1 X control value). Uremic animals were pair-fed with sham-operated controls. In the proximal tibia delayed transformation of cartilage into primary spongiosa with appearance of chondro-osteoid and delayed transformation of primary spongiosa into secondary spongiosa was observed (rickets). Increased amounts of osteoid were present although 25-OH-vitamin D3-levels were high. There were only modest signs of secondary hyperparathyroidism (osteoclast counts; urinary cAMP). In spite of the presence of bone disease, longitudinal growth was not reduced in uremic animals as compared with pair-fed sham-operated animals, but was significantly reduced as compared with ad lib fed control animals. In contrast, weight gain was significantly diminished in uremic animals as compared with pair-fed sham-operated control animals. It is concluded that diminished intake of food is the major determinant of growth retardation in preterminal experimental renal failure.

Detection of colorectal neoplasms by the highly sensitive hemoglobin-haptoglobin complex in feces.

Abstract Screening for fecal occult blood by means of guaiac tests has an unsatisfactory sensitivity for the detection of colorectal neoplasms. The immunological determination of human hemoglobin in feces has a higher sensitivity and specificity, but hemoglobin is degraded during its transport through the gastrointestinal tract. We compared the hemoglobin test to a newly developed immuno-chemiluminometric (ILMA) assay for quantifying the hemoglobin-haptoglobin complex in feces which shows high stability against degradation. From each of 621 patients with gastrointestinal complaints before scheduled colonoscopy we collected two 1-ml samples from a single stool; there were no dietary restrictions. The sensitivity for detecting colorectal carcinomas proved 87% with hemoglobin. With the hemoglobin-haptoglobin complex it was 87% at a cutoff level of 1.5 µg/g feces, 83% at 2.0 µg/g feces, and 78% at 2.5 and 3.0 µg/g feces. The sensitivity for detecting large adenomatous polyps was 54% with hemoglobin, 76% with the hemoglobin-haptoglobin complex at a cutoff point of 1.5 µg/g feces, 73% with the hemoglobin-haptoglobin complex at 2.0 and 2.5 µg/g feces, and 65% with the hemoglobin-haptoglobin complex at 3.0 µg/g feces. The optimal cutoff point for the hemoglobin-haptoglobin complex was estimated to be 2.0 µg/g stool. The specificity for hemoglobin (99%) was significantly higher than that for the hemoglobin-haptoglobin complex at 2.0 µg/g feces (96%). Immunological determination of the hemoglobin-haptoglobin complex in feces has a comparable sensitivity as the fecal hemoglobin assay for colorectal carcinomas and a significantly higher sensitivity for adenomatous polyps but a significantly lower specificity. Its use for colorectal cancer prevention is currently being evaluated in a screening study.

Vascular calcifications under maintenance hemodialysis

ZusammenfassungIn einer Querschnittsstudie wurden 101 erwachsene Hämodialysepatienten röntgenologisch (Beckenübersicht, Vorfuß) untersucht. Gefäßverkalkungen (Vorfuß) wurden bei 20% aller Dialysepatienten gefunden. Diese Zahl liegt wesentlich niedriger als frühere Angaben. Die Häufigkeit von Gefäßverkalkungen war höher bei Patienten, deren Dialysebeginn mehrere Jahre zurücklag. In einer prospektiven Verlaufskontrolle wurde Neuauftreten von Gefäßverkalkung jedoch nur bei einem von 50 Patienten beobachtet, obwohl Hyperparathyreoidismus und mäßiggradige Hyperphosphatämie unter der Hämodialyse persistierten. Bei urämischen Kindern (56 urämische Kinder ohne Hämodialyse, 82 urämische Kinder mit Hämodialyse) wurden Gefäßverkalkungen nur in einem einzigen Falle gefunden. Hingegen wurde in 3 von 11 Kindern viscerale Verkalkungen des Lungengerüstes bei der Autopsie gefunden.SummaryIn a cross-sectional study X-rays of the forefoot and the pelvis of 101 adult dialysis patients were taken. Vascular calcifications (forefoot) were observed in 20 patients. The incidence was higher in patients who had been started on dialysis several years ago. However, in a longitudinal prospective study de novo appearance of vascular calcifications was observed only in 1 out of 50 dialysed patients, although hyperparathyreoidism and moderate hyperphosphatemia persisted. Vascular calcifications were seen only once in 138 uremic children (56 uremic children without dialysis; 82 uremic children on maintenance hemodialysis). However at autopsy visceral calcifications of the lung were found in three (out of 11) children who did not have vascular calcifications on X-rays.

Disturbed calcium metabolism in subjects with elevated diastolic blood pressure

SummaryEssential hypertension has been associated with disturbed calcium metabolism, but the available data are controversial. We measured parameters of calcium metabolism in groups of untreated male subjects (n = 78) with elevated diastolic blood pressure (101 ± 6 mmHg, mean ± SD) and age-matched male subjects (n=79) with low diastolic blood pressure (62 ± 4 mmHg). The participants of the study were drawn from a random population sample. Subjects with high diastolic blood pressure had significantly higher carboxy-terminal parathyroid hormone (PTH) plasma concentrations than controls with low diastolic blood pressure (median 114 vs. 43 pmol/l, P < 0.01). The 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D concentrations were comparable in both groups. Individuals with high diastolic blood pressure had significantly lower total serum calcium (2.41 ± 0.10 vs. 2.47 ± 0.10 mmol/l, mean ± SD; P < 0.01). PTH concentrations were correlated with diastolic pressure (r = −0.39, P < 0.001). The data are compatible with increased parathyroid activity despite unchanged concentrations of vitamin D metabolites in human hypertension.