A. Pilo

The Disposal of an Oral Glucose Load in Healthy Subjects: A Quantitative Study

Although it is an established concept that the liver is important in the dispositionof glucose, the quantitative contribution of the splanchnic and peripheral tissues, respectively,to the disposal of an oral glucose load is still controversial. In the present investigation, we have employed the hepatic venous catheter technique in combination with a double-tracer approach(in which the glucose pool is labeled with 3H-glucose and the oral glucose load is labeled with 14C-glucose) to quantitate the four determinants of oral glucose tolerance: rate of oral glucose appearance, splanchnic glucose uptake, peripheral glucose uptake, and suppression of hepatic glucose production. Studies were carried out in 11 normal volunteers in the overnight-fasted state and for 3.5 h after the ingestion of glucose (1 g/kg body wt; range, 55–93 g). In the postabsorptive state, the rate of endogenous (hepatic) glucose production, evaluated from the 3H-glucose infusion, was 2.34 ± 0.06 mg/min · kg. Glucose ingestion was accompanied by a prompt reduction of endogenous glucose output, which reached a nadir of 0.62 ± 0.23 mg/min kg at 45 min and remained suppressed after 3.5 h (0.85 ±0.22 mg/ min · kg). The average inhibition of hepatic glucose output during the absorptive period was 53 ± 5%. The appearance of ingested glucose in arterial blood, as derived from the 14C-glucose measurements after correction for recycling 14C radioactivity, reached a peak after 15–30 min, and 14C-glucose continued to enter the systemic circulation throughout the observation period. The rate of appearance of ingested glucose was 2.47 ± 0.45 mg/min kg at 3.5 h. A total of 73 ± 4% of the oral load was recovered in the systemic circulation within 3.5 h. The cumulative net output of glucose from the splanchnic area, measured directly with the hepatic vein catheter technique, was 46 ± 5 g over 3.5 h. This net splanchnic glucose balance resulted from the appearance of 50 ± 5 g of the glucose load plus a residual hepatic production of 15 ± 2 g, minus a splanchnic glucose uptake of 19 ± 4 g. Splanchnic fractional extraction of glucose (basal = 2.7 ± 0.7%) failed to increase in response to glucose ingestion. Splanchnic glucose uptake, however, was significantly (P < 0.001) higher during the absorptive period (19 ± 4 g/3.5 h) than in the basal state (5 ± 1 g/3.5 h). Peripheral glucose uptake (48 ± 6 g/3.5 h) was also enhanced by glucose ingestion (P < 0.001 versus a basal value of 27 ± 2 g/3.5 h) and accounted for over 70% of total glucose disposal. It is concluded that, after the ingestion of a glucose load in healthy subjects: (1) endogenous glucose production is suppressed by approximately 50%, (2) both splanchnic and peripheral uptake of glucose are stimulated, (3) the rise in splanchnic uptake is achieved primarily by augmented glucose availability rather than by increased splanchnic fractional extraction of glucose, and (4) peripheral glucose uptake accounts for the majority Of total glucose disposal.

Kinetic Analysis of Plasma Insulin Disappearance in Nonketotic Diabetic Patients and in Normal Subjects: A TRACER STUDY WITH 125I-INSULIN

The studies so far reported on the metabolic clearance rate of insulin in human diabetes mellitus have given conflicting results, probably because they have been conducted on few patients and have used a variety of experimental techniques and data treatments. We investigated the kinetics of insulin distribution and degradation in 35 normal subjects and in 42 nonketotic, nonobese, overtly diabetic patients, of whom 26 were above 40 yr old and 16 were 40 yr old or less at diagnosis. The design of the study combined (a) the use of a tracer to perturb minimally the steady state and to avoid glucose infusion; (b) the preparation of purified [(125)I]-monoiodoinsulin, which has a metabolic behavior similar to that of native insulin; and (c) noncompartmental analysis of the plasma immunoprecipitable (125)I-insulin disappearance curves, which were recorded for 2 h after pulse i.v. injection of the tracer.Metabolic clearance rate was found to be similar in diabetics (404+/-18 ml/min.m(2), mean+/-SEM) and in normals (420+/-14), although the latter-onset patients had slightly, if not significantly, lower metabolic clearance rate values than the earlier-onset diabetics (385+/-19 and 443+/-36, respectively). The initial distribution volume of the hormone also did not significantly differ in diabetics and normals and was similar to plasma volume. The reentry rate into the initial distribution volume of the hormone and the total, plasma-equivalent distribution volume of insulin were both significantly raised in diabetics (251+/-12 ml/min.m(2) and 10.3+/-0.5 liters/m(2)) in comparison with normals (195+/-8 and 7.5+/-0.3). The posthepatic delivery rate of insulin was found to be slightly raised in later-onset diabetics (194+/-20 mU/h.m(2)), but somewhat reduced in earlier-onset diabetics (133+/-15) in comparison with normals (172+/-14); these differences reflected the different basal plasma insulin concentrations in these three groups. Chronic treatment with oral hypoglycemic drugs, age, duration of the disease, and degree of metabolic control appeared to have only little effect on the kinetics of insulin.On the basis of these results, we conclude that insulin-independent adult diabetics show, already in the fasting state, a combination of insulin resistance and insulin deficiency and a derangement in insulin distribution, the precise significance of which is uncertain.

Proficiency testing project for brain natriuretic peptide (BNP) and the N-terminal part of the propeptide of BNP (NT-proBNP) immunoassays: the CardioOrmocheck study.

Abstract Background: We organized and conducted a proficiency testing study (CardioOrmocheck) to evaluate the differences in analytical performance of brain natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) immunoassays. Methods: Approximately 90 Italian laboratories were involved in the 2005–2007 proficiency testing cycles, while 112 laboratories took part in the 2008 cycle (from January to May 2008). A total of 28 study samples were measured by participating laboratories for a total of 2354 determinations. Results: The mean total variability for BNP (50.6 %CV) was significantly higher than that for NT-proBNP (8.4 %CV). In addition, the mean variability due to differences between-methods (46.4 %CV) comprised the majority of the total variability for BNP. Between-method variability for BNP comprised, on average, 84% of total variability, while the within-method variability comprised an average of 20.2 %CV. On the contrary, for NT-proBNP the within-method variability (7.3 %CV) represented the majority of total variability (average 75%), while between-method variability was smaller (4.1 %CV). Imprecision around the cut-off value showed marked differences among methods, especially for BNP immunoassay methods. In addition, BNP methods were affected by large systematic differences, for example an average 2.7-fold difference between Access and ADVIA Centaur methods, while agreement between NT-proBNP methods was better (an average 1.2-fold difference between Dimension and ECLIA on the Elecsys methods). Conclusions: This multicenter collaborative study demonstrates that there are significant differences in analytical characteristics and measured values among the most popular commercial methods for BNP and NT-proBNP. Clinicians should be very careful when comparing results obtained by laboratories that use different methods. Clin Chem Lab Med 2009;47:762–8.

Clinical relevance of biological variation: the lesson of brain natriuretic peptide (BNP) and NT-proBNP assay

Abstract The clinical relevance of brain natriuretic peptide (BNP) and N-terminal (NT)-proBNP assays as a diagnostic tool and prognostic marker in patients with cardiovascular diseases has recently been confirmed. However, several studies demonstrated variation of intra-individual BNP concentrations of >30% (ranging from 30% to 50%) with reference change values at the 95% confidence interval (i.e., the estimated critical difference) ranging from 99% to 130% in healthy subjects and heart failure patients. According to this estimated confidence interval, only a great variation in plasma BNP levels should be considered significant in an individual patient (for example, a decrease of >50% or an increase of more than two-fold). Many recent clinical studies have demonstrated that BNP variations below this estimated critical difference could also have clinical relevance. Like the concentration of other neuro-hormones, levels of plasma BNP fluctuate widely and rapidly along with heart rhythm and blood pressure variations in response to physiological stimuli. However, biological variation of BNP should not be interpreted strictly as random fluctuation around a homeostatic set point, as assumed by the common model used in all studies on biological variation of BNP reported in the literature. These results cannot be directly transferred to clinical practice. While awaiting more accurate studies, we suggest that variations of plasma BNP three-fold greater than the analytical imprecision should be considered as potentially relevant from a physiological and clinical point of view.

Altered Tissue Degradation and Distribution of Atrial Natriuretic Peptide in Patients With Idiopathic Dilated Cardiomyopathy and Its Relationship With Clinical Severity of the Disease and Sodium Handling

BACKGROUND Atrial natriuretic peptide (ANP) has been suggested to play an important role in heart failure, preserving cardiorenal homeostasis through maintenance of the sodium balance and inhibition of the detrimental effects of the neurohormonal vasoconstrictor system. The current study was designed to investigate whether there is a disturbed renewal and distribution of ANP in patients with idiopathic dilated cardiomyopathy (IDC) with differing clinical severity of disease. METHODS AND RESULTS We used a tracer method to perform a cross-sectional study of 15 IDC patients with differing clinical severity (New York Heart Association functional class I to III), prospectively divided into two groups according to their functional class (group 1, classes I and II; group 2, classes II-III and III). Eleven normotensive, nonobese male volunteers also were studied as a control group. Main ANP kinetic parameters were derived from the disappearance curve of the labeled hormone after the bolus injection of [125I]-labeled ANP. A high-performance liquid chromatography technique was used to separate the radiolabeled hormone in each plasma sample. Patients in group 1 showed higher ANP metabolic clearance rate (MCR) (2731.9 +/- 726.2 mL.min-1.m-2) than patients of group 2 (1718.4 +/- 621.2 mL.min-1.m-2) and control subjects (1873.1 +/- 551.2 mL.min-1.m-2). ANP disposal (MCR) positively correlated with biological hormonal effect (urinary sodium excretion) both in control subjects and in patients. In IDC patients of both groups, however, MCR values were always higher (approximately doubled) than the values found in control subjects at the corresponding sodium excretion. This finding indicates that a reduced ANP biological activity is associated with hormone degradation in patients. Moreover, patients of group 2 showed significantly higher ANP production rates (395.6 +/- 183.8 ng.min-1.m-2) than group 1 (166.0 +/- 139.0 ng.min-1.m-2) and control subjects (130.7 +/- 105.4 ng.min-1.m-2) despite a marked reduction in sodium excretion. Patients with IDC showed a progressive reduction in the total distribution volume (group 1, 19.8 +/- 5.8 L/m2; group 2, 12.7 +/- 6.9 L/m2; control subjects, 27.0 +/- 11.6 L/m2) of the hormone; this probably was due to a reduction in exchanges of ANP with peripheral tissues. CONCLUSIONS Our study demonstrates a markedly altered degradation and distribution of ANP in patients with IDC, even in those at the early stage of clinical disease (classes I and II, group 1) who have ANP plasma levels in the normal range.

Turnover Studies on Cardiac Natriuretic Peptides: Methodological,Pathophysiological and Therapeutical Considerations.

Cardiac natriuretic peptide hormones (ANP and BNP) are synthesized and secreted by the heart, producing several biological effects, such as natriuresis, vasorelaxation, hypotension, and neuromodulation. Extensive studies conducted in both animals and humans have documented that cardiac natriuretic peptides (CNPs) are secreted into the circulatory system via the coronary sinus into the right atrium, and then rapidly degraded and removed from the blood by plasma proteases and specific clearance receptors. Usually, studies of CNPs kinetics have been carried out following an experimental protocol in which labeled or unlabeled hormone is administered (by constant infusion or bolus injection) and the corresponding concentration of the hormone is measured in peripheral venous blood. However, when a uniform intravascular concentration throughout artero-venous vessels is lacking due to the very rapid clearance of the substance being studied (such as CNPs), the classical compartmental or none compartmental approach may not be suitable for interpreting the experimental data. In this case, a more physiological circulatory model, which does not assume a uniform intravascular distribution of the hormone and comprises several anatomo-functional blocks arranged in a series and supplied by the same flow (cardiac output) should be adopted. Different experimental designs (infusion or bolus injection) as well as multiple sampling sites (aorta and pulmonary artery, inferior vena cava, femoral vein) were used in ANP kinetic studies. Using a circulatory approach, ANP has been demonstrated to be rapidly distributed and degraded; in healthy subjects about 50% of ANP secreted into the right atrium is extracted by the peripheral tissues during the first pass throughout the body. Since CNPs have important fluid-volume regulatory features, it has been postulated that they also play a key role in volume homeostasis in several pathophysiological states, such as congestive heart failure. Indeed, a markedly altered degradation and distribution of ANP in patients with cardiac failure who show a resistance to its natriuretic effects, even in those on the early stage of clinical disease, whose CNPs plasma levels are in the normal range, have been demonstrated. Recent studies indicate that some drugs, by inhibiting the degradation of CNPs by plasma proteases and can thus affect CNP kinetics, may be useful in the treatment of arterial hypertension and cardiac failure.

Uric acid metabolism in normal subjects and in gouty patients by chromatographic measurement of 14C-uric acid in plasma and urine.

The turnover kinetics of uric acid were determined in a group of 25 subjects, including 6 controls, 17 gouty patients, and 2 subjects with asymptomatic hyperuricemia. According to their daily urate excretion while on a purine-free diet, the gouty patients (none of whom had evidence of tophi at the time of the study) were classified as follows: five overexcretor patients (“metabolic” gout), six normo-excretor patients (“renal” gout), and six borderline patients. Uric acid-2-14C was used as the tracer, and the uric acid radioactivity in plasma and urine samples was measured after column chromatography through a polyacrylamide gel that specifically adsorbs uric acid. The experimental data of plasma disappearance curves and urine radioactivities of 14C-uric acid (followed up to 3 days after injection) were analyzed by the noncompartmental approach and by the urine/plasma ratio method, in order to calculate the total removal rate of uric acid, its distribution volumes, and the removal rate of uric acid through the renal route. The monocompartmental approach and the urine specific radioactivity methods were also employed to compute the uric acid turnover data, and the results were compared with those of noncompartmental analysis. The overexcretor patients were found to have normal metabolic clearance rate and fractional catabolic rate values, but significantly higher-than-normal total turnover rate values (p < 0.005), whereas the normo-excretors had normal total turnover rate values but significantly lower metabolic clearance rate and fractional catabolic rate values compared to the controls (p < 0.001). Uric acid excretion through the kidneys was found to be about two-thirds of the total removal both in the control subjects and in the gouty patients. The results obtained allowed the exclusion of any significant delay in the excretion of uric acid through the kidneys. The use of monocompartmental analysis led to a significant overestimation of both the total turnover rate (p < 0.005) and the total pool of uric acid (p < 0.001), and the urine specific radioactivity approach implied a significant overestimation of the total turnover rate (p < 0.001). The gouty patients as a whole had a significantly larger-than-normal (p < 0.001) total pool of uric acid, however determined. On the basis of the results obtained, a metabolic differentiation was made possible for the two pathophysiologic types of gout. In fact, the metabolic gout patients were all characterized by normal metabolic clearance rate values and increased total pool values, whereas the renal gout patients were all characterized by reduced metabolic clearance rate values and increased total pool values.

Insulin Degradation In Vitro and In Vivo: A Comparative Study in Men: Evidence That Immunoprecipitable, Partially Rebindable Degradation Products Are Released From Cells and Circulate in Blood

The products of insulin metabolism generated in vitro and in vivo were compared in this study. Monocytes from 10 control subjects were incubated with 125IA14-labeled insulin, acid washed, and solubilized or reincubated in insulin-free binding buffer to study both intracellular radioactivity or radioactivity released from cells to medium. To evaluate in vivo insulin metabolism, labeled insulin (100–120 μCi) was injected as a single intravenous bolus in 5 of the 10 subjects. Cellular and plasma radioactivity was characterized by high-performance liquid chromatography (HPLC). The results of the study show the following: 1) Products with superimposable HPLC elution profiles are found within cells and in medium. Two new labeled products are observed in the latter, suggesting that a membrane degradation process exists in monocytes. 2) Intermediates found within monocytes, in medium from monocytes, and in plasma have identical elution profiles, supporting the possibility that insulin is metabolized in various cells by a common pathway. 3) Insulin metabolism produces intermediates that bind well to anti-insulin antibody. The presence in plasma of these products induces a significant difference in the value of the metabolic clearance rate of insulin when HPLC or immunoprecipitation is used to detect intact insulin. 4) Immunoprecipitable products maintain, in part, the capacity to bind to insulin receptors and to be internalized into monocytes.

Comparison of immunoassays for tumor markers CA 19-9, CA 15-3 and CA 125: data from an international quality assessment scheme.

Data collected in the 1993 and 1994 cycles of an international external quality assessment (EQA) program and in a national multicenter collaborative study were cumulatively analyzed to evaluate the standardization of the methods currently in use for the assay of mucinous tumor markers CA 19-9, CA 15-3 and CA 125. On average the between-laboratory variability was 15.2 and 16.0 CV% for CA 15-3 and CA 125 respectively; the between-laboratory variability found for CA 19-9 was markedly worse (mean 28.3 CV%). The variability component attributable to systematic differences between different methods/kits was relatively small for CA 15-3 and CA 125 (18% and 24% of the total variability) but markedly larger for CA 19-9 (48% of the total variability). The agreement of CA 19-9 results worsened in the last few years when new nonisotopic techniques became available. The precision of the methods/kits most used in the survey ranged from 9.9 to 13.3 CV% for CA 125 and from 11.6 to 13.9 CV% for CA 15-3. For these two tumor markers the precision of the traditional IRMAs does not appear different from that of the new fully automated nonisotopic techniques. The precision of CA 19-9 methods was on average worse (from 11.7 to 19.6 CV%) although two automated systems exhibited a precision better than that of IRMAs. In conclusion, the results of this study indicate that CA 15-3 and CA 125 are satisfactorily assayed whereas CA 19-9 assay appears affected by larger differences between methods and by poorer precision of laboratories and kits.

Role of serum carrier proteins in the peripheral metabolism and tissue distribution of thyroid hormones in familial dysalbuminemic hyperthyroxinemia and congenital elevation of thyroxine-binding globulin.

To investigate the role of thyroxine-binding globulin (TBG) and albumin in the availability of thyroid hormones to peripheral tissues, comprehensive kinetic studies of thyroxine (T4) and triiodothyronine (T3) were carried out in eight subjects with familial dysalbuminemic hyperthyroxinemia (FDH), in four subjects with inherited TBG excess, and in 15 normals. In high-TBG subjects, the reduction of T4 and T3 plasma clearance rates (by 51% and 54%, respectively) was associated with normal daily productions; T4 and T3 distribution volumes were significantly reduced. In FDH subjects T4 clearance was less reduced (by 31%) than in high TBG; consequently T4 production rate was significantly increased (by 42%); T4 and T3 distribution volumes and T3 clearance rate were unchanged. Increased T3 peripheral production in FDH (by 24%) indicates that T4 bound to abnormal albumin is more available to tissues than T4 carried by TBG, thus suggesting an important role of albumin in T4 availability to the periphery.