Zoledronic acid and alendronate sodium and the implications in orthodontic movement.

OBJECTIVE: To evaluate orthodontic tooth movement (OTM) in rats treated with two types of bisphosphonates (BPs), alendronate sodium (A) and zoledronic acid (Z). DESIGN: In all, 15 male Wistar rats were randomly divided into three groups. Group OTM+A: orthodontic tooth movement and subcutaneous administration of alendronate sodium (2.5 mg/kg); Group OTM+Z: orthodontic tooth movement and subcutaneous administration of zoledronic acid (0.02 mg/kg), and Group OTM: orthodontic tooth movement and subcutaneous injection of saline. The BPs were administered once a day during 25 days before OTM started and during 10 days of OTM. The left upper first molar was moved with a stainless-steel closed coil spring which delivered an initial force of 0.4N. OTM was measured with a digital caliper comparing the moved and the contralateral side. The histomorphometric analysis counted the number of osteoclasts, inflammatory cells, blood vessels and fibroblasts (n/104  m2 ) in periodontal ligament (PDL) of the distobuccal root. RESULTS: A reduction of 58.3% of OTM was found in Group OTM+A and 99.6% in Group OTM+Z, when compared with Group OTM. There was a significant decrease of osteoclasts and inflammatory cells in BP-treated groups. Blood vessels and fibroblastic cells decreased mainly in Group OTM+Z. CONCLUSION: Alendronate sodium and zoledronic acid have similar effects on the periodontal tissue during orthodontic treatment in rats. Especially, zoledronic acid can affect orthodontic tooth movement.

Structure and viscoelastic behavior of pharmaceutical biocompatible anionic microemulsions containing the antitumoral drug compound doxorubicin.

Structure and viscoelastic properties of negatively charged oil-in-water (o/w) microemulsions have been investigated. Microemulsions (ME) containing soya phosphatidylcholine (SPC), eumulgin HRE 40 (EU) and sodium oleate (SO) as surfactant, cholesterol (CHO) as oil phase, and aqueous buffer with and without the antitumoral doxorubicin (DOX) have been studied. The effect of the oil phase/surfactant ratio (O/S) and the DOX incorporation on the structural and rheological properties have been studied in several compositions of ME systems. The rheological analyses were performed through the oscillation stress sweep, creep recovery test, and viscosity test. The combination of the DOX incorporation with the high O/S ratio provided a further viscoelastic structure with linear behavior. Independently of the O/S ratio the oil phase diameter increases according to a sigmoid profile, stabilizing up to 340 min. The apparent viscosity decreases a minimum value with the shear rate, but increases with both the O/S ratio and the DOX incorporation in the system. The structural and rheological properties of the studied MEs were directly dependent on the O/S ratio and can be used to improve the application of the system in the pharmaceutical field.

Oil-in-water lecithin-based microemulsions as a potential delivery system for amphotericin B.

In this work the structural features of microemulsions (MEs) containing the pharmaceutical biocompatible Soya phosphatidylcholine/Tween 20 (1:1) as surfactant (S), Captex 200 as oil phase (O), and phosphate buffer 10mM, pH 7.2 as aqueous phase (W) were studied. Systems obtained with different proportions of the components were described by pseudo-ternary phase diagrams in order to characterize the microemulsions studied here. MEs were prepared with and without the polyene antifungal drug amphotericin B (AmB). The maximum AmB incorporation into the ME system was dependent on both the oil phase and surfactant proportions with 6.80 and 5.7 mg/mL in high contents, respectively. The incorporation of AmB into the ME systems significantly increased the profile of the droplet size of the ME for all ranges of surfactant proportions used in the formulations. The microstructures of the system were characterized by dynamic light scattering (DLS) and rheological behavior. The DLS results showed that the size of the oil droplets increases 4.6-fold when AmB is incorporated into the ME system. In all cases the increase in the proportion of the oil phase of the ME leads to a slight increase in the diameter of the oil droplets of the system. Furthermore, for both the AmB-loaded and AmB-unloaded MEs, the size of the oil droplets decrease significantly with the increase of the S proportion in the formulations, demonstrating the efficiency of the surfactant in stabilizing the ME. Depending on the ME composition, an anti-thixotropic behavior was found. The maximum increases of the consistency index caused by the increase of the oil phase of the ME were of 17- and 25-times for the drug-loaded and drug-unloaded MEs, respectively. However, the observed effect for the drug-loaded ME was about 4.6 times higher than that for the drug-unloaded one, demonstrating the strong effect of the drug on the rheological characteristics of the ME system. Therefore, it is possible to conclude that the investigated ME can be used as a very promising vehicle for AmB.

Structural changes of biocompatible neutral microemulsions stabilized by mixed surfactant containing soya phosphatidylcholine and their relationship with doxorubicin release.

Depending on the composition, the mixture of surfactant, oil and water, may form supramolecular aggregates with different structures which can significantly influence the drug release. In this work several microemulsion (ME) systems containing soya phosphatidylcholine (SPC) and eumulgin HRE40 (EU) as surfactant, cholesterol (O) as oil phase, and ultra-pure water as an aqueous phase were studied. MEs with and without the antitumoral drug doxorubicin (DOX) were prepared. The microstructures of the systems were characterized by photon correlation spectroscopy, rheological behavior, polarized light microscopy, small-angle X-ray scattering (SAXS) and X-ray diffraction (XRD). The results reveal that the diameter of the oil droplets was dependent on the surfactant (S) amount added to formulations. The apparent viscosity was dependent on the O/S ratio. High O/S ratio leads to the crystallization of cholesterol polymorphs phases which restricts the mobility of the DOX molecules into the ME structure. Droplets with short-range spatial correlation were formed from the ME with the low O/S ratio. The increase of the cholesterol fraction in the O/S mixture leads to the formation of ordered structures with lamellar arrangements. These different structural organizations directly influenced the drug release profiles. The in vitro release assay showed that the increase of the O/S ratio in the formulations inhibited the constant rate of DOX release. Since the DOX release ratio was directly dependent on the ratio of O/S following an exponential decay profile, this feature can be used to control the DOX release from the ME formulations.

High pressure liquid chromatography and electrospray ionization mass spectrometry are advantageously integrated into a two-levels approach to detection and identification of haemoglobin variants.

Detecting and correctly identifying haemoglobin (Hb) variants is typically achieved by a two-levels laboratory approach. We report our experience in dealing with 91 Hb variants, including a number of frequent and a few rare variants. Screening included akaline agarose gel electrophoresis (AGE), ion-exchange automated high-performance liquid chromatography (HPLC) and a test for deoxyhaemoglobin solubility. Identification was based on electrospray ionization-mass spectrometry (ESI-MS). Our results confirmed the advantages of HPLC over AGE for screening, because of the occurrence of some electrophoretically 'silent' variants. ESI-MS permitted the definitive identification of 90 of the 91 variants included in the study, in some cases (e.g. HbS) through the application of a simple protocol (direct injection of the sample), in other cases requiring the application of more demanding procedures (purification of the variant chain and peptide analysis after enzymatic or chemical cleavage). In an additional case (Hb J-Oxford), ESI-MS assay did not lead to definitive identification, but gave indications for designing the appropriate primers to focus DNA sequence analysis on the specific region of the gene. Deoxyhaemoglobin solubility test was positive only in the presence of HbS. We conclude that HPLC and ESI-MS are advantageously integrated into a two-level analytical system for the detection and confirmation of variant Hbs.

B-type natriuretic peptide in athletes performing an Olympic triathlon.

AIM: Brain natriuretic peptide (BNP) is a cardioactive molecule produced in the myocardium. BNP is a sensitive marker of cardiac failure and its measurement in blood could be useful to the diagnosis and the treatment of this disease. Sporting activities, especially endurance ones, can induce cardiac problems, owing to the high workload for the myocardium during long and ultralong heavy effort. There are 2 papers describing the behavior of BNP in endurance events. BNP was elevated in marathoners, immediately after the race and also after 4 h. We studied the behavior of BNP in the triathlon, which is a complex sport characterized by 3 different activities (swimming, cycling, running). METHODS: We recruited 49 athletes, all males, except for 4 females; 2 athletes did not finish the race and were not included in the statistical analysis in 2 different competitions. In these subjects we measured BNP using an immunological method before and after a triathlon. RESULTS: No statistical significance between BNP values, before and after the triathlon, was found. CONCLUSIONS: We found no significant differences between pre- and postcompetition BNP values. Moreover, the range of values in both the blood drawings are similar of those of the general population, representing the biological variability of the analyte. The values in regularly trained athletes,, are not different from the general population and BNP is not modified by a triathlon, a typical endurance sport performance. We can underline that BNP increases in plasma are induced by heavy pathologies and are not influenced by physical activities, even strenuous ones.

The size of pancreatic pseudocyst does not influence the outcome of invasive treatments.

BACKGROUND: Authors generally agree that Giant Pancreatic Pseudocysts (> 10 cm) have a lower spontaneous resolution and are more difficult to treat than smaller pancreatic pseudocysts. This study was carried out on two groups of patients with larger and smaller pancreatic pseudocysts (pancreatic pseudocysts > 10 cm versus pancreatic pseudocysts < 10 cm), and aims to establish whether the size of pancreatic pseudocysts is a factor influencing treatment outcomes. PATIENTS AND METHODS: In a retrospective study, we examined 71 patients with pancreatic pseudocysts following an episode of acute pancreatitis, which were treated in our hospital from 1980 to 2000. Forty-one (57.5%) patients had a large pancreatic pseudocyst. Most patients underwent invasive treatments: 9 (12.6%) had percutaneous drainage, 37 (52.1%) open surgery and 13 (18.3%) endoscopic cyst gastrostomy. 12 patients (16.9%) of the 71 were cured with medical therapy alone. RESULTS: As far as the aetiology of the pancreatitis, location and number of the cysts were concerned, no major differences emerged between the two groups, although large pancreatic pseudocysts followed more severe pancreatitis (P = 0.0005). All giant pancreatic pseudocysts required invasive treatments; 40% of the pancreatic pseudocysts < 10 cm were successfully treated with medical therapy alone. No statistical differences were found regarding hospital mortality, morbidity, recurrence rate and hospital stay among the patients treated invasively. CONCLUSIONS: Giant pancreatic pseudocysts more often require invasive therapy due to persistent symptoms or complications. Treatment outcomes do not seem to be influenced by the size of the pancreatic pseudocysts.

Low-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol and apolipoprotein B: relationships among the different measurements.

Serum low-density lipoprotein cholesterol (LDLC) value is a recognized target for atherosclerotic risk management, and is generally calculated using the "Friedewald formula". Alternative risk markers include directly measured LDLC, non-high-density lipoprotein cholesterol (non-HDLC) and apolipoprotein B (ApoB). The relationships among such various measured or calculated quantities in medium-sized sets of patient results were investigated. Results from two sets of patients were retrieved from our laboratory information systems. One group (n=8436) included results of cholesterol, HDLC, triglyceride (TG) and glucose measurements. A second group (n = 902) included, in addition, results of ApoB measurement. The results confirmed the unreliability of the Friedewald formula at TG >350 mg/dL (3.96 mmol/L), but also indicated TG-linked underestimation of LDLC below such a TG level. By contrast, non-HDLC values were shown to be independent of TG, and better correlated to ApoB than LDLC values. Mathematically, the difference between non-HDLC and LDLC is TG x 0.458 (values in mmol/L): therefore, the latter cannot be compared to (or converted into) the former by simply adding a constant amount. The ratio LDLC/ApoB was shown continuously to decrease with increasing TG concentrations, while the ratio non-HDLC/ApoB did not. The TG-dependent underestimation of LDLC may be the reason for the reported better cardiovascular risk predictivity of non-HDLC in diseases associated with TG increase, such as in diabetes. Non-HDLC values are not influenced by TG levels, and are better correlated with ApoB.

Commutability of calibration and control materials for serum lipase.

BACKGROUND: To effectively assess and correct for intermethod variability, calibration and control materials (CCMs) must show the same intermethod behavior as patient sera, i.e., they must be commutable. We describe the commutability of selected CCMs for lipase assays, the impact of noncommutability of CCMs in normalizing patient results, and characteristics of reagents that affect assay specificity and commutability. METHODS: Lipase was measured in 98 patient sera and in 29 commercial CCMs, with 2 commercial methods using different substrates and with 4 experimental methods using 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester as substrate and colipase as cofactor, but differing in the stabilizing proteins used and in the size of the substrate micelles. RESULTS: The noncommutability rate, i.e., the frequency of aberrant intermethod behavior of CCMs in comparison with patient sera, was 27% for liquid CCMs and 47% for lyophilized CCMs. The normalized residuals, measuring the degree of noncommutability, were -2.3 to 2.4 for CCMs with "normal" lipase activity, and -3.5 to 21.7 for CCMs with higher lipase activity. Recalculation of patient results with CCMs as calibrators decreased or increased the original bias according to whether the CCMs were commutable. CONCLUSIONS: For the lipase methods in this study, the frequency of noncommutability of CCMs is affected by assay-specific characteristics, including size of substrate micelles and the presence or absence of added proteins.

Central and baroreflex control of heart rate during the wake-sleep cycle in rat.

UNLABELLED: Spontaneous fluctuations in Heart Period (HP) and Mean Arterial Pressure (MAP) make it possible to evaluate baroreceptor-heart rate reflex sensitivity (BRS). 30-s sequences of HP and MAP beat-to-beat values were considered in the different wake-sleep states (Wake, W; Quiet Sleep, QS; Active Sleep, AS) in rats to assess whether 1) BRS changes between states and 2) the different indexes supply consistent BRS measures. BRS indexes were calculated according to validated literature procedures as regression coefficients of HP vs. MAP 1) within all ramps of increasing or decreasing MAP of four beats or more, with HP and MAP changing in the same direction (baroreflex-mediated fluctuations, BRSp), 2) within all such ramps irrespective of the relative direction of HP and MAP changes (baroreflex + non-baroreflex, i.e. non-homeostatic centrally driven, fluctuations, BRSA). HP vs. MAP regression coefficient along the entire 30-s sequence (bHPMAP) was also calculated. RESULTS: BRSp did not change among states, BRSA decreased from QS to W to AS, bHPMAP decreased from QS to W and became negative in AS. CONCLUSIONS: 1) as indicated by BRSp, baroreflex sensitivity is state independent, 2) BRSp to BRS(A) to bHPMAP are increasingly affected by non-baroreflex fluctuations, BRSp being most apt to measure BRS, 3) non-homeostatic MAP and HP fluctuations increase from QS to W and prevail in AS. These potentially harmful fluctuations are normally buffered by baroreflexes: in the case of baroreflex impairment, circulatory risk may arise in conditions like AS, when they prevail.

Myoglobin and creatine kinase isoenzyme MB mass assays: intermethod behaviour of patient sera and commercially available control materials.

The low biological variation of myoglobin and creatine kinase isoenzyme MB mass (CK-MBm) requires accurate measurements. In the standardization process, in order to effectively measure and correct intermethod variability, the intermethod behaviour of control materials must be the same of patient sera, i.e. they must be commutable. In this work we checked the commutability of some commercially available control materials in pairs of methods for myoglobin and CK-MBm measurements; we assessed the impact of commutable and non-commutable control materials when used for equalizing patient sera results by two different methods and discussed the problems related to external quality assessment schemes. Myoglobin and CK-MBm were measured in sets of 49 and 56 patient sera and in 13 commercially available control materials with two automatic analytical systems. The non-commutability rate was 8.3% for myoglobin and 23.1% for CK-MBm. Recalculation of serum samples results with a control material as calibrator lowered or increased the bias originally present according to whether the material itself was commutable or not. We conclude that also for myoglobin and CK-MBm assays it is necessary to check the commutability of materials to be used in external quality assessment schemes, or to normalize patient results by different methods.

FESCC survey on accreditation and post-graduate training in clinical chemistry in European countries. Federation of European Societies of Clinical Chemistry.

Promotion of the professional growth and development of specialists in the field of clinical chemistry in European countries, and harmonisation of quality assessment and accreditation procedures are listed among the main goals and activities of Federation of European Societies of Clinical Chemistry (FESCC), according to its 1999-2000 strategic plan. The European countries that are members of the European Union are in the process of establishing the "European Register for Clinical Chemists", based on minimum standards of education, training and experience as defined by the European Communities Confederation of Clinical Chemists (EC4). Many other European countries would like to adapt their system of professional education to this model. Data on post-graduate training in EC4 FESCC members have already been gathered in 1998. However, at the present time, there is no detailed knowledge of pre- and post-graduate professional education of specialists in clinical chemistry in the non-EC4 European countries. FESCC launched a survey in July 1998 in order to gather this information with the hope to start a database about existing systems. All FESCC members received the same questionnaire on accreditation (seven questions) and non-EC4 FESCC members received an additional questionnaire with 11 questions related to post-graduate training in clinical chemistry. The response rate of the 35 FESCC member countries was 93% from the 15 EC4 members (14 responses/15 countries) and 80% from the 20-non-EC4 (16 responses/20 countries). The heterogeneity of the data on post-graduate training in clinical chemistry indicates that a great effort will be needed before harmonisation is reached. These results, however, will provide an interesting basis for further discussion and promotion of post-graduate training in clinical chemistry. The data provided on accreditation show that the total number of accredited laboratories was relatively low in EC4 countries and even lower in non-EC4 members. It was not surprising to see that the number of accredited laboratories was the highest in the two countries which started accreditation the earliest (i.e. Sweden and UK, 1992). This situation, however, is changing at a fast rate in most countries and the number of the accredited sites is expected to increase rapidly in the next few years.

Low frequency of elevated serum transferrin saturation in elderly subjects.

Serum transferrin saturation (TS) values were calculated on the basis of serum iron and transferrin (protein) measurements in a total of 2425 serum samples from six groups of subjects: individuals applying for selection as blood donors (M and F, median age 34 and 32 years); patients referring to the hospital laboratory for routine testing (M and F, median age 45 and 48 years); and elderly subjects living in a specialized institute (M and F, median age 76 and 82 years). In the first four groups the frequency of TS values <15% and >62% respectively, was substantially as expected, considering the average health conditions and sex. These results indirectly support the reliability of the measurement procedure. In the elderly group, however, the frequency of TS values >62% was zero. Mean TS values in the elderly group (males and females) were significantly lower (P<0. 0001) than in the blood donors group and in the hospital patients one. This observation suggests a shortened survival in the presence of (unrecognized) iron overload, pointing out at the usefulness of iron overload screening using simple biochemical tests.

Density of perfused brain capillaries in the aged rat during the wake-sleep cycle.

Structural and functional age-related changes in brain vasculature might affect the cerebral microcirculation. The present study evaluated the density of perfused brain capillaries and the perfusion fraction (perfused/existing capillaries) in aged rats (24 months) during the states of the wake-sleep cycle (quiet wakefulness, quiet sleep, active sleep) characterized by different levels of brain activation. The number of perfused capillaries was determined by intravascular injection of the fluorescent marker Evans Blue; histochemical staining of the capillary endothelium identified the alkaline-phosphatase (AP) reactive quota of the anatomical population. No sleep-related changes in perfused capillary density were found, and the perfusion fraction in the AP-stained sub-population was high and stable (95%) across the sleep-wake cycle: changing levels of brain activation during sleep do not affect functional capillary density in aged rats.

Cerebral circulation in REM sleep: is oxygen a main regulating factor?

In the transition from NREM to REM sleep, as in other instances of brain activation, a marked increase in cerebral blood flow and glucose uptake is observed, together with a lesser increase in O2 uptake. Brain activation also entails an increase in capillary PO2 and lactate production. The hypothesis of saturation of the oxidative machinery was advanced to explain anaerobic glycolysis and lactate production in the presence of high PO2, but data are available that cannot be explained by this hypothesis: hypoxic spots exist in the brain, augmenting in arterial hypoxia and disappearing in arterial hyperoxia, while tissue [H+] lowers as arterial PO2 increases beyond 100 mmHg. Additional hypotheses are thus required. We suggest that O2 diffusion limitation exists in the brain: microregions lying at mid-distance between capillaries may become hypoxic and partly resort to anaerobic glycolysis. These microregions are thought to enlarge with increasing metabolic rate or arterial hypoxia and give rise to vasodilatatory signals regulating local blood flow. REM sleep time is strongly reduced by hypoxic and increased by hyperoxic atmosphere, in accordance with the existence of an O2 diffusion limitation. Any pathological decrease in arterial PO2 and/or O2 delivery creates a specific risk in REM sleep.

Cerebral blood flow regulation in REM sleep: a model for flow-metabolism coupling.

The pattern of metabolic and circulatory changes occurring during REM sleep in the whole brain is also observed at a regional level in different instances of functional activation. This pattern is characterized by an increase in metabolic rate, blood flow, glucose and oxygen uptake, the increase in glucose uptake generally exceeding oxygen uptake. A model of interpretation is presented, based on the assumption that substantial limitation to oxygen diffusion exists in the brain. According to the model, microregions lying at mid-distance between capillaries may become hypoxic, depending on metabolic rate and blood-cell PO2 difference. At increasing metabolic rates, O2 consumption in pericapillary microregions increases and the PO2 drop becomes steeper. As a consequence, in microregions far from capillaries a decrease in O2 availability occurs, in concomitance with the increase in metabolic rate, so that non-oxidative glucose metabolism develops locally. A similar spatial PO2 pattern forms in the case of arterial hypoxia, when capillary PO2, and then blood-cell PO2 difference, is reduced. The hypoxic microregions are the source of vasodilatatory messages, the consequent vasodilatation increasing average capillary PO2 and then favoring O2 diffusion to the tissue. Oxygen thus appears to be a better candidate than glucose as a mediator of blood flow-metabolism coupling. This is supported by its higher extraction fraction and by the fact that, in physiologic conditions, arterial hypoxia (and not hypoglycemia) acts on cerebral blood flow. Moreover, the diffusion capacity of glucose in the brain is higher than that of oxygen, so that diffusion limitation is more likely to occur for oxygen. The present model allows consistent organization of the stereotyped changes in cerebral blood flow and glucose and oxygen uptake occurring both in REM sleep and in other instances of brain activation.

Serum proteins by capillary zone electrophoresis: approaches to the definition of reference values.

The Paragon CZE 2000 (Beckman Analytical, Milan, Italy) is an automatic dedicated capillary zone electrophoresis (CZE) system, producing a five-zone serum protein pattern with quantitative estimation of the zones. With the view of substituting this instrument for two previously used serum protein electrophoresis techniques, we planned to produce reference values for the "new" systems leading to compatible interpretation of the results. High resolution cellulose acetate electrophoresis with visual inspection and descriptive reporting (HR-CAE) and five-zone cellulose acetate electrophoresis with densitometry (CAE-D) were the previously used techniques. Serum samples (n = 167) giving "normal pattern" with HR-CAE were assayed with the CZE system, and the results were statistically assessed to yield 0.95 reference intervals. One thousand normal and pathological serum samples were then assayed with the CAE-D and the CZE techniques, and the regression equations of the CAE-D values over the CZE values for the five zones were used to transform the CAE-D reference limits into the CZE reference limits. The two sets of reference values thereby produced were in good agreement with each other and also with reference values previously reported for the CZE system. Thus, reference values for the CZE techniques permit interpretation of results coherent with the previously used techniques and reporting modes.