Synthetic Hct Research Articles

Estimating synthetic hematocrit and extracellular volume from native blood pool T1 times at 3 Tesla CMR: Derivation of a conversion equation, accuracy and comparison with published formulas

PurposeCalculation of extracellular volume fraction (ECV), a marker of myocardial fibrosis in cardiac magnetic resonance imaging (CMR), requires hematocrit (Hct). We aimed to correlate Hct levels with native blood T1 times, to derive a formula for estimating synthetic Hct (Hctsyn) and synthetic ECV (ECVsyn), to assess accuracy of ECVsyn and to compare our model with published formulas. MethodIn this retrospective study, a cohort of 250 CMR scans with T1 mapping (3T, MOLLI 5(3)3, endsystolic aquisition), was divided into a derivation and validation cohort. Native T1 times of the left ventricular blood pool (T1native,midLV) were correlated with Hct levels from blood sampling within 24 h (Hct24h) and a formula for calculation of Hctsyn was derived by linear regression. ResultsIn the derivation cohort (n = 167), Hct24h showed a good association with T1native,midLV (r = −0.711, p < 0.001). The resulting regression equation was Hctsyn = 1/T1native,midLV * 1355.52–0.310. In the validation cohort (n = 83), Hctsyn and Hct24h showed good correlation (r = 0.726, p < 0.001), while ECVsyn, and ECV24h demonstrated excellent correlation (r = 0.940, p < 0.001). ECVsyn had a minimal bias of 0.28 % and the misclassification rate (8.8 %) was comparable to the variability introduced by repeated Hct measurements (misclassification in 7.5 %). Applying published formulas in our cohort resulted in incorrect classification in up to 60 %. ConclusionWe provide a formula for estimating Hctsyn from native blood T1 on a 3T scanner. The measurement error of ECVsyn is low and comparable to the error due to retest variability of conventional Hct. Scanner- and sequence-specific formulas should be used.

Native T1 mapping and extracellular volume fraction for differentiation of myocardial diseases from normal CMR controls in routine clinical practice

BackgroundThis study aimed to determine native T1 and extracellular volume fraction (ECV) in distinct types of myocardial disease, including amyloidosis, dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), myocarditis and coronary artery disease (CAD), compared to controls.Methods We retrospectively enrolled patients with distinct types of myocardial disease, CAD patients, and control group (no known heart disease and negative CMR study) who underwent 3.0 Tesla CMR with routine T1 mapping. The region of interest (ROI) was drawn in the myocardium of the mid left ventricular (LV) short axis slice and at the interventricular septum of mid LV slice. ECV was calculated by actual hematocrit (Hct) and synthetic Hct. T1 mapping and ECV was compared between myocardial disease and controls, and between CAD and controls. Diagnostic yield and cut-off values were assessed.ResultsA total of 1188 patients were enrolled. The average T1 values in the control group were 1304 ± 42 ms at septum, and 1294 ± 37 ms at mid LV slice. The average T1 values in patients with myocardial disease and CAD were significantly higher than in controls (1441 ± 72, 1349 ± 59, 1345 ± 59, 1355 ± 56, and 1328 ± 54 ms for septum of amyloidosis, DCM, HCM, myocarditis, and CAD). Native T1 of the mid LV level and ECV at septum and mid LV with actual and synthetic Hct of patients with myocardial disease or CAD were significantly higher than in controls.ConclusionsAlthough native T1 and ECV of patients with cardiomyopathy and CAD were significantly higher than controls, the values overlapped. The greatest clinical utilization was found for the amyloidosis group.

Myocardial extracellular volume fraction quantification in an animal model of the doxorubicin-induced myocardial fibrosis: a synthetic hematocrit method using 3T cardiac magnetic resonance.

Visualization of diffuse myocardial fibrosis is challenging and mainly relies on histology. Cardiac magnetic resonance (CMR), which uses extracellular contrast agents, is a rapidly developing technique for measuring the extracellular volume (ECV). The objective of this study was to evaluate the feasibility of the synthetic myocardial ECV fraction based on 3.0 T CMR compared with the conventional ECV fraction. This study was approved by the local animal care and ethics committee. Fifteen beagle models with diffuse myocardial fibrosis, including 12 experimental and three control subjects, were generated by injecting doxorubicin 30 mg/m2 intravenously every three weeks for 24 weeks. Short-axis (SAX) and 4-chamber long-axis (LAX) T1 maps were acquired for both groups. The association between hematocrit (Hct) and native T1blood was derived from 9 non-contrast CMR T1 maps of 3 control beagles using regression analysis. Synthetic ECV was then calculated using the synthetic Hct and compared with conventional ECV at baseline and the 16th and 24th week after doxorubicin administration. The collagen volume fraction (CVF) value was measured on digital biopsy samples. Bland-Altman plots were used to analyze the agreement between conventional and synthetic ECV. Correlation analyses were performed to explore the association among conventional ECV, synthetic ECV, CVF, and left ventricular ejection fraction (LVEF). The regression model synthetic Hct = 816.46*R1blood - 0.01 (R2=0.617; P=0.012) was used to predict the Hct from native T1blood values. The conventional and synthetic ECV fractions of experimental animals at the 16th and 24th week after modeling were significantly higher than those measured at the baseline (31.4%±2.2% and 36.3%±2.1% vs. 22.9%±1.7%; 29.9%±2.4% and 36.1%±2.6% vs. 22.0%±2.4%; all with P<0.05). Bland-Altman plots showed a bias (1.0%) between conventional and synthetic ECV with 95% limits of agreement of -2.5% to 4.4% in the per-subject analysis (n=21) and a bias (1.0%) between conventional and synthetic ECV with 95% limits of agreement of -2.4% to 4.3% in the per-segment analysis (n=294). Conventional and synthetic ECV were well correlated with CVF (r=0.937 and 0.925, all with P<0.001, n=10). Our study showed promising results for using synthetic ECV compared with the conventional ECV for providing accurate quantification of myocardial ECV without the need for blood sampling.

Is a timely assessment of the hematocrit necessary for cardiovascular magnetic resonance\u2013derived extracellular volume measurements?

BackgroundCardiovascular magnetic resonance (CMR)–derived extracellular volume (ECV) requires a hematocrit (Hct) to correct contrast volume distributions in blood. However, the timely assessment of Hct can be challenging and has limited the routine clinical application of ECV. The goal of the present study was to evaluate whether ECV measurements lead to significant error if a venous Hct was unavailable on the day of CMR.Methods109 patients with CMR T1 mapping and two venous Hcts (Hct0: a Hct from the day of CMR, and Hct1: a Hct from a different day) were retrospectively identified. A synthetic Hct (Hctsyn) derived from native blood T1 was also assessed. The study used two different ECV methods, (1) a conventional method in which ECV was estimated from native and postcontrast T1 maps using a region-based method, and (2) an inline method in which ECV was directly measured from inline ECV mapping. ECVs measured with Hct0, Hct1, and Hctsyn were compared for each method, and the reference ECV (ECV0) was defined using the Hct0. The error between synthetic (ECVsyn) and ECV0was analyzed for the two ECV methods.ResultsECV measured using Hct1 and Hctsyn were significantly correlated with ECV0 for each method. No significant differences were observed between ECV0 and ECV measured with Hct1 (ECV1; 28.4 ± 6.6% vs. 28.3 ± 6.1%, p = 0.789) and between ECV0 and ECV calculated with Hctsyn (ECVsyn; 28.4 ± 6.6% vs. 28.2 ± 6.2%, p = 0.45) using the conventional method. Similarly, ECV0 was not significantly different from ECV1 (28.5 ± 6.7% vs. 28.5 ± 6.2, p = 0.801) and ECVsyn (28.5 ± 6.7% vs. 28.4 ± 6.0, p = 0.974) using inline method. ECVsyn values revealed relatively large discrepancies in patients with lower Hcts compared with those with higher Hcts.ConclusionsVenous Hcts measured on a different day from that of the CMR examination can still be used to measure ECV. ECVsyn can provide an alternative method to quantify ECV without needing a blood sample, but significant ECV errors occur in patients with severe anemia.

P5281Synthetic extracellular volume quantification by cardiac magnetic resonance imaging - a reliable tool in clinical practice?

Abstract Background Extracellular volume (ECV) quantification by CMR imaging allows the measurement of changes in the extracellular space indicating diffuse edema and fibrosis, especially in early stages of myocardial disease. Usually venous hematocrit (Hct) is needed in order to calculate standard ECV. Recent findings suggest the alternative use of the blood pool T1-relaxation time (T1blood) instead of the venous Hct to generate “synthetic Hct ” and furthermore “synthetic ECV”, allowing a more routine and user-friendly application without the need of a current blood sample. Purpose Purpose of this study is to re-evaluate whether the connection between venous Hct and T1blood can be used to generate a synthetic ECV in a large number of volunteers and patients with various myocardial diseases from a tertiary care centre. Methods 945 volunteers and patients were divided into a derivation (n=490) and a validation group (n=455). Standard ECV was calculated using T1-relaxtion times acquisitioned in the septal myocardium and blood pool, each on pre- and post-contrast Images using a modified look-locker sequence at a 3 Tesla MR-scanner. In addition venous Hct was measured in a blood sample drawn prior to the scan. Synthetic Hct was derived through linear regression analysis between venous Hct and 1/T1blood and was then compared to venous Hct. Also we derived standard ECV and synthetic ECV and examined these using Lin's rho and Bland-Altman comparison. Results In the derivation group venous Hct and 1/T1blood showed a linear relationship (R2=0,22; p=ehz746.02521). This was used to calculate synthetic Hct and synthetic ECV. Synthetic ECV correlated well with standard ECV (Lin's rho= 0,903; p&lt;0,0001). Mean difference 0,004, limits of agreement −0,038 and 0,047. See table 1 for patient characteristics and figure 1 for Bland-Altman plot on standard and synthetic ECV. Table 1. Patient characteristics Sex female (%) 312 (33%) Age, yrs 60±15 EDVi, ml/m2 86±31 ESVi, ml/m2 43±30 LVEF, % 54±16 Hematocrit, % 43±5 Figure 1 Conclusion Despite only weak correlation of synthetic and venous Hct we found excellent correlation in synthetic and standard ECV. Therefore we could confirm the value of synthetic ECV in a large cohort of volunteers and patients, providing a feasible and non-invasive marker for extracellular myocardial alterations in everyday clinical practice.

Automatic quantification of the myocardial extracellular volume by cardiac computed tomography: Synthetic ECV by CCT

BackgroundThe quantification of extracellular volume fraction (ECV) by Cardiac Computed Tomography (CCT) can identify changes in the myocardial interstitium due to fibrosis or infiltration. Current methodologies require laboratory blood hematocrit (Hct) measurement – which complicates the technique. The attenuation of blood (HUblood) is known to change with anemia. We hypothesized that the relationship between Hct and HUblood could be calibrated to rapidly generate a synthetic ECV without formally measuring Hct. MethodsThe association between Hct and HUblood was derived from forty non-contrast thoracic CT scans using regression analysis. Synthetic Hct was then used to calculate synthetic ECV, and in turn compared with ECV using blood Hct in a validation cohort with mild interstitial expansion due to fibrosis (aortic stenosis, n = 28, ECVCT = 28 ± 4%) and severe interstitial expansion due to amyloidosis (n = 27; ECVCT = 54 ± 11%, p < 0.001). For histological validation, synthetic ECV was correlated with collagen volume fraction (CVF) in a separate cohort with aortic stenosis (n = 18). All CT scans were performed at 120 kV and 160 mAs. ResultsHUblood was a good predictor of Hct (R2 = 0.47; p < 0.01), with the regression model (Hct = [0.51 * HUblood] + 17.4) describing the association. Synthetic ECV correlated well with conventional ECV (R2 = 0.96; p < 0.01) with minimal bias and 2SD difference of 5.7%. Synthetic ECV correlated as well as conventional ECV with histological CVF (both R2 = 0.50, p < 0.01). Finally, we implemented an automatic ECV plug-in for offline analysis. ConclusionSynthetic ECV by CCT provides instantaneous quantification of the myocardial extracellular space without the need for blood sampling.

Synthetic hematocrit derived from the longitudinal relaxation of blood can lead to clinically significant errors in measurement of extracellular volume fraction in pediatric and young adult patients

BackgroundExtracellular volume fraction (ECV) is altered in pathological cardiac remodeling and predicts death and arrhythmia. ECV can be quantified using cardiovascular magnetic resonance (CMR) T1 mapping but calculation requires a measured hematocrit (Hct). The longitudinal relaxation of blood has been used in adults to generate a synthetic Hct (estimate of true Hct) but has not been validated in pediatric populations.MethodsOne hundred fourteen children and young adults underwent a total of 163 CMRs with T1 mapping. The majority of subjects had a measured Hct the same day (N = 146). Native and post-contrast T1 were determined in blood pool, septum, and free wall of mid-LV, avoiding areas of late gadolinium enhancement. Synthetic Hct and ECV were calculated and intraclass correlation coefficient (ICC) and linear regression were used to compare measured and synthetic values.ResultsThe mean age was 16.4 ± 6.4 years and mean left ventricular ejection fraction was 59% ± 9%. The mean measured Hct was 41.8 ± 3.0% compared to the mean synthetic Hct of 43.2% ± 2.9% (p < 0.001, ICC 0.46 [0.27, 0.52]) with the previously published model and 41.8% ± 1.4% (p < 0.001, ICC 0.28 [0.13, 0. 42]) with the locally-derived model. Mean measured mid-free wall ECV was 30.5% ± 4.8% and mean synthetic mid-free wall ECV of local model was 29.7% ± 4.6% (p < 0.001, ICC 0.93 [0.91, 0.95]). Correlations were not affected by heart rate and did not significantly differ in subpopulation analysis. While the ICC was strong, differences between measured and synthetic ECV ranged from −8.4% to 4.3% in the septum and −12.6% to 15.8% in the free wall. Using our laboratory’s normal cut-off of 28.5%, 59 patients (37%) were miscategorized (53 false negatives, 6 false positives) with published model ECV. The local model had 37 miscategorizations (20 false negatives, 17 false positives), significantly fewer but still a substantial number (23%).ConclusionsOur data suggest that use of synthetic Hct for the calculation of ECV results in miscategorization of individual patients. This difference may be less significant once synthetic ECV is calculated and averaged over a large research cohort, making it potentially useful as a research tool. However, we recommend formal measurement of Hct in children and young adults for clinical CMRs.

Automatic Measurement of the Myocardial Interstitium: Synthetic Extracellular Volume Quantification Without Hematocrit Sampling

ObjectivesThe authors sought to generate a synthetic extracellular volume fraction (ECV) from the relationship between hematocrit and longitudinal relaxation rate of blood. BackgroundECV quantification by cardiac magnetic resonance (CMR) measures diagnostically and prognostically relevant changes in the extracellular space. Current methodologies require blood hematocrit (Hct) measurement—a complication to easy clinical application. We hypothesized that the relationship between Hct and longitudinal relaxation rate of blood (R1 = 1/T1blood) could be calibrated and used to generate a synthetic ECV without Hct that was valid, user-friendly, and prognostic. MethodsProof-of-concept: 427 subjects with a wide range of health and disease were divided into derivation (n = 214) and validation (n = 213) cohorts. Histology cohort: 18 patients with severe aortic stenosis with histology obtained during valve replacement. Outcome cohort: For comparison with external outcome data, we applied synthetic ECV to 1,172 consecutive patients (median follow-up 1.7 years; 74 deaths). All underwent CMR scanning at 1.5-T with ECV calculation from pre- and post-contrast T1 (blood and myocardium) and venous Hct. ResultsProof-of-concept: In the derivation cohort, native R1blood and Hct showed a linear relationship (R2 = 0.51; p < 0.001), which was used to create synthetic Hct and ECV. Synthetic ECV correlated well with conventional ECV (R2 = 0.97; p < 0.001) without bias. These results were maintained in the validation cohort. Histology cohort: Synthetic and conventional ECV both correlated well with collagen volume fraction measured from histology (R2 = 0.61 and 0.69, both p < 0.001) with no statistical difference (p = 0.70). Outcome cohort: Synthetic ECV related to all-cause mortality (hazard ratio 1.90; 95% confidence interval 1.55 to 2.31; for every 5% increase in ECV). Finally, we engineered a synthetic ECV tool, generating automatic ECV maps during image acquisition. ConclusionsSynthetic ECV provides validated noninvasive quantification of the myocardial extracellular space without blood sampling and is associated with cardiovascular outcomes.

Human Calcitonin Tetrameric Gene: Comparative Expression in Yeast and Transgenic Potato Plants

ABSTRACTHuman calcitonin (hCT) is a 32 amino acid peptide participating in the regulation of calcium and phosphorus metabolism in humans. It is used in clinics for treatment of diseases related to bone decalcification, such as Paget disease, osteoporosis imperfecta, parathyroid gland carcinomas, etc. A synthetic hCT tetrameric (hCTt) gene is now cloned in binary vectors for gene expression in yeast and plants. The vector for expression in yeast is based on 2μ plasmid containing the α-factor leader sequence. The plasmid for expression in plants carries the 35S cauliflower mosaic virus promoter linked to the tobacco etch virus 5′-nontranslated leader sequence acting as a translational enhancer. Two strains of yeast cells (GRF18 and VY168) and one cultivar of potato (Solanum tuberosum Nevski) were transformed and expression of the hCTt gene was studied by mRNA-DNA hybridization, RT-PCR, Northern-blot analysis, ELISA and RIA. The yield of recombinant hCTt was estimated to be about 12–20 mg per liter of yeast culture for yeast and 0.02% of the total soluble protein in transgenic potato plants.

Efficacy of intranasal human calcitonin in patients with Paget's disease refractory to salmon calcitonin

purpose: A cause for the resistance to intranasal salmon calcitonin (sCT) therapy in patients with Paget's disease is the occurrence of neutralizing antibodies to sCT. As a result, a new formulation of intranasal human calcitonin (hCT) was developed, and the efficacy investigated in patients treated earlier with sCT. patients and methods: Twelve patients with Paget's disease were treated twice daily for 6 months with 1 mg synthetic hCT administered intranasally. Five patients demonstrated low-titer antibodies to sCT, and four of the patients did not respond previously to 1-year therapy with intranasal sCT. The hypocalcemic effect of 3 mg hCT was compared to that of 0.1 mg sCT before and after the intranasal hCT therapy. Serum alkaline phosphatase and the ratio between the urinary excretion of hydroxyproline and creatinine were measured before and during intranasal hCT treatment. results: The hypocalcemic response to 3 mg intranasal hCT (−6.60 ± 0.67%, mean ± standard error) was similar before and at the end of intranasal hCT therapy (−5.92 ± 0.80%, p >0.1). Intranasal sCT (0.1 mg) lowered serum calcium less effectively (−2.86 ± 0.76%) than 3 mg intranasal hCT (p >0.05). The presence of low-titer antibodies to sCT did not affect the hypocalcemic response to sCT or hCT. As a result of the 6-month intranasal hCT regimen, serum alkaline phosphatase and urinary hydroxyproline/creatinine ratio were reduced to 62 − 5% (p <0.001) and 80 ± 7% (p <0.05), respectively, or pretreatment levels. In four patients previously resistant to intranasal sCT therapy because of neutralizing antibodies to sCT, serum alkaline phosphatase was similarly lowered by intranasal hCT to 66 ± 6% of pretreatment levels (p <0.05). conclusion: A new formulation of intranasal hCT effectively lowered serum calcium levels, alkaline phosphatase concentrations, and urinary hydroxyproline excretion in patients with Paget's disease, some of whom were previously resistant to intranasal sCT because of neutralizing antibodies.

Calcitonin Determination by a Fast and Highly Sensitive Enzyme Amplified Immunoassay

A colorimetric enzyme amplification system was used to develop an immunoassay for human calcitonin (hCT) with a sensitivity of 6 pmol/l, and intra- and inter-assay CVs of 12% and 11.8% respectively for the low pool, and 10% and 11.2% for the high pool. The mean recovery of added synthetic hCT (58.5 pmol) from the plasma of 10 patients was 110% (64.4 pmol). The correlation coefficient between radioimmunoassay (RIA) and amplified enzymo-immunoassay was found to be 0.96 (p 0.001). The assay was successfully applied to the measurement of elevated calcitonin levels in plasma from patients with medullary carcinoma of the thyroid (MCT). AEIA offered a reliable and sensitive alternative to RIA for calcitonin determination with the added advantage of convenience as the label employed was much more stable.

Radioimmunoassay of urinary calcitonin in normal subjects and in patients with medullary carcinoma of the thyroid.

A simple technique for measuring urine calcitonin (CT) was established using a sensitive radioimmunoassay (RIA) system for plasma human calcitonin (hCT). To extract urinary CT, urine samples were fractioned by gel chromatography on a column (0.8 x 20 cm) of Bio Gel P-2. Recovery of synthetic (1-32)hCT was 86.1 +/- 6.2% and the intra- and inter-assay coefficients of variation in RIA were 5.9 and 8.2%, respectively. Dilution curves of the urinary CT after gel-filtration were parallel with the standard curve. In 11 patients with medullary carcinoma of the thyroid (MCT), the CT levels of the urine (in ng/mg Cr) were 3.4 to 20.8 times higher than those of the plasma (in ng/ml), and a significant positive correlation (r = 0.93, P less than 0.001) was obtained between the urinary and plasma levels of CT in these 11 patients. In 32 normal subjects, the CT levels of the urine (in pg/mg Cr) were only 0.41 to 5.1 times of those of the plasma (in pg/ml), and a weak positive correlation (r = 0.408, P less than 0.05) was obtained between these two levels. However, urinary CT level in normal subjects apparently reflected a rise in endogenous plasma hCT in response to calcium infusion (4 mg/kg B.W. for 1 min) and that of exogenous plasma CT when synthetic (1-32)hCT (500 micrograms for 1 min) was injected intravenously. Following gel filtration on a Bio Gel P-30 1.5 x 80 cm column, larger molecular forms of CT than (1-32)hCT were found in the urine not only patients with MCT but also of normal subjects after infusion of calcium or synthetic (1-32)hCT. It is concluded that measurement of urine CT is useful not only for diagnosis of MCT but also for evaluating CT metabolism in normal subjects.

Serum and urine calcitonin levels in patients with medullary thyroid carcinoma and with non-thyroidal cancer, and their immunological size-heterogeneity (author's transl)

Human calcitonin (hCT) levels were measured in serum and urine samples from normal subjects and patients with medullary thyroid calcinoma (MCT) and non-thyroidal cancer (NTC). The immunological size-heterogeneity of calcitonin was examined in some of these samples.The hCT level in serum was measured directly by RIA and that in urine was measured after purification; that is, 2 ml of urine sample were boiled for 2 min, mixed with blue dextran and passed through a Bio Gel P-2 0.8 × 20 cm column, and the blue void volume was used for measurement of hCT.The hCT levels in the serum of 66 normal subjects ranged from less than 25 to 140 pg/ml, and those in the urine of 56 normal subjects ranged from 66 to 270 pg/mg creatinine (Cr). The hCT levels in the serum of 24 patients with MTC ranged from 0.24 to 215 ng/ml, and those in the urine of 11 of the 24 ranged from 1.7 to 520 ng/mgCr. In the 11 patients with MTC, the hCT levels in the urine (in ng/mgCr) were 3.4 to 20.8 times higher than those in the serum (in ng/ml), and these two levels were positively correlated with each other (r=0.93, p<0.001). The hCT levels in the serum of 277 patients with NTC ranged from less than 25 to 2013 pg/ml, and those of 44 patients (16%) with various kinds of non-thyroidal cancer were abnormally high (>150 pg/ml). The urine hCT levels of 82 of the 277 patients ranged from 60 to 83,000 pg/mgCr, and the levels of 16 patients (20%) were abnormally high (>300 pg/mgCr). In one patient with lung cancer, the serum and urine hCT levels were both abnormally high (1.8 ng/ml and 80 ng/mgCr, respectively), and an oat cell carcinoma of the lung was found at autopsy.On gel filtration on a Bio Gel P-30 1.5 × 80 cm column, four distinct forms of immunoreactive calcitonin were observed in serum samples from three patients with MTC; one was eluted in the same position as synthetic hCT and the other three in positions corresponding to higher molecular weights. The main form in urine samples from these three patients was eluted in a position corresponding to one form with higher molecular weight in the serum, not in the position of synthetic hCT. The serum sample from the patient with oat cell carcinoma of the lung contained three bigger forms of immunoreactive calcitonin, and no form eluted with synthetic hCT. A urine sample from this patient also contained one main form which was eluted as a bigger form similar to that of MTC patients.It was concluded that the measurement of urine hCT levels is useful not only for detecting cases of MTC but also of ectopic calcitonin secreting tumors, and that the urine of these patients contains a specific form that is bigger than synthetic hCT.

Effect of acute and chronic administration of calcitonin on serum glucose in patients with Paget's disease of bone.

The effects of an acute injection of synthetic salmon calcitonin (sCT) and human CT (hCT) and of long term (4-month) administration of sCT on serum glucose levels were investigated in eight patients with Paget's disease of bone. The results obtained demonstrate a small but statistically significant rise in serum glucose after a single sc injection of synthetic hCT. However, the serum glucose level was not increased after 4 months of daily administration of synthetic sCT to our pagetic patients. Our results also substantiate the clinical observation that long term administration of CT does not cause clinical diabetes or significantly change fasting blood glucose concentration. Our results are also consistent with the view that the effect of CT administration on glucose metabolism is related to the secondary hypocalcemia.

Radioimmunoassay of human calcitonin using labeled [Asu1, 7]-human calcitonin analogue.

Application of synthetic human calcitonin analogue ([Asul,7]-hCT) was attempted, since it is chemically more stable than native hCT. [Asu1, 7]-hCT was successfully labeled with 125I to a specific activity ranging from 410 to 540 microCi/microgram. Sensitivity, recovery and reproducibility of the assay system using 125I-labeled [Asu1,7]-hCT analogue, standard synthetic hCT and its antibodies, were 49 pg/ml, 99.8 +/- 4%, and 7.95% (C. V. within assay) or 21.9% (C. V. between assays), respectively. The dilution curve of the sera obtained from patients with medullary carcinoma paralled well with the standard curve. 125-I-labeled [Asu1, 7]-hCT analogue can be used for routine radioimmunoassay of hCT.

Synthetic human calcitonin: analysis of antibodies obtained from various animal species and determination of immunoreactive hormone in human sera.

Antibodies to synthetic human calcitonin (hCT) were developed in rabbits, goats and mice. The free peptide (32 amino-acid residues, Mwt. 3418) was administered together with adjuvant, and the effect of various immunization procedures, as well as of different dose-levels, was evaluated comparatively. Synthetic hCT was found to be a good immunogen for the three animal species examined. The relative importance of various structural parts of the hCT molecule with regard to immunological specificity was determined by reference to the inhibition of the specific binding of 125I-hCT to antibodies by peptide fragments of hCT. All the antisera studied were directed to structural and/or conformational properties of the 11-28 or 11-32 amino acid sequence of hCT. Six different antisera from rabbits and goats were selected for radioimmunological assay of hCT on the basis of their inhibitory dose50-values and immunological specificity. To improve the sensitivity of the radioimmunoassay (RIA), we studied the preparation of radioiodinated hCT and assessed various parameters determining the sensitivity of the assay. Despite all the efforts, CT in human plasma from healthy subjects could not be determined with certainty. The difficulties encountered in the determination of normal levels of circulating CT are discussed in terms of the sensitivity of RIA and non-specific interference of serum factors with RIA.