The ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) K121Q polymorphism modulates the beneficial effect of weight loss on fasting glucose in non-diabetic individuals.

BACKGROUND AND AIMS: Several studies have reported that the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) K121Q polymorphism (rs1044498) interacts with increased adiposity in affecting glucose homeostasis and insulin sensitivity. Conversely, one would expect that the amelioration of glucose homeostasis observed after weight loss is modulated by the ENPP1 K121Q polymorphism. The aim of our study was to test such hypothesis, in non-diabetic overweight-obese individuals. METHODS AND RESULTS: Two hundred eleven non-diabetic overweight-obese individuals were studied. Body mass index (BMI), fasting glucose, homeostasis model assessment of insulin resistance (HOMA-IR index) and lipid levels were obtained before and after 6-week lifestyle intervention (LI; diet and exercise) and their changes calculated as baseline minus 6-week values. LI decreased BMI, glucose, HOMA-IR and triglyceride levels (p < 0.001 for all). No difference across genotype groups (160 KK and 51 KQ or QQ - named as XQ - individuals) was observed in these changes. In a multivariate model, BMI changes predicted fasting glucose changes (ß = 0.139 mmol/L (2.50 mg/dl) for 1 unit BMI change, p = 0.005). This correlation was not significant among KK individuals (ß = 0.082; p = 0.15), while much steeper and highly significant among XQ individuals (ß = 0.336; p = 0.00008) (p-value for Q121-by-weight loss interaction = 0.047). CONCLUSION: Individuals carrying the ENPP1 Q121 variant are highly responsive to the effect of weight loss on fasting glucose. This reinforces the previously suggested hypothesis that the Q121 variant interacts with adiposity in modulating glucose homeostasis.

Vitamin D and parameters of calcium homeostasis in inpatients with and without Type 2 diabetes mellitus.

AIM: We investigated inpatients with and without Type 2 diabetes mellitus, aged over 60 yr, to compare their vitamin D status and calcium homeostatic parameters. MATERIALS AND METHODS: We studied 140 patients consecutively admitted to our Internal Medicine Unit during the year 2010 (61 from November to April, 79 from May to October). The sample encompassed 70 patients with and 70 without diabetes. At admission we measured serum calcium (Ca), phosphate (P), sodium (Na), potassium (K), creatinine (Cr), alkaline phosphatase total activity (AP), albumin adjusted serum calcium (Caalb adj), 25 hydroxy-vitamin D (25OHD), PTH, and 24-h urinary Na/Cr (uNa/Cr), K/Cr (uK/Cr), Ca/Cr (uCa/Cr), P/Cr (uP/Cr) ratios, and calcium excretion (Ca ex). RESULTS: 25OHD levels of patients with and without diabetes did not significantly differ. In patients without diabetes recruited from November to April, 25OHD levels were significantly lower than those from May to October, whilst patients with diabetes did not show a significant seasonal variation. PTH had opposite non-significant seasonal variations, and negatively correlated with 25OHD in both groups of patients. This correlation was lost after adjusting for age and body mass index in patients with diabetes. These inpatients had higher serum P and lower uP/Cr, according to lower PTH. Their serum glucose negatively correlated with uCa/Cr and Ca ex, contrary to inpatients with other diseases. Instead, uCa/Cr and Ca ex correlated with uNa/Cr only in patients without diabetes. CONCLUSIONS: Inpatients with diabetes did differ from those with other disorders for vitamin D status and calcium-phosphate homeostatic mechanism.

SARS-CoV-2 was already circulating in Italy, in early December 2019.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) identified in China, in December 2019 determines COronaVIrus Disease 19 (COVID-19). Whether or not the virus was present in Italy earlier the first autochthonous COVID-19 case was diagnosed is still uncertain. We aimed to identify anti-SARS-CoV-2 antibodies in sera collected from 4th November 2019 to 9th March 2020, in order to assess the possible spread of the virus in Italy earlier than the first official national diagnosis. PATIENTS AND METHODS: Anti-SARS-CoV-2 antibodies were evaluated in retrospective serum samples from 234 patients with liver diseases (Hep-patients) and from 56 blood donors (BDs). We used two rapid serologic tests which were confirmed by a validated chemoluminescence assay. RESULTS: Via rapid tests, we found 10/234 (4.3%) IgG-positive and 1/234 (0.4%) IgM-positive cases in the Hep-patient group. Two/56 (3.6%) IgG-positive and 2/56 (3.6%) IgM-positive cases were detected in BD group. Chemoluminescence confirmed IgG-positivity in 3 Hep-patients and 1 BD and IgM-positivity in 1 Hep-patient. RNAemia was not detected in any of the subjects, rendering the risk of transfusion transmission negligible. CONCLUSIONS: Our results suggest an early circulation of SARS-CoV-2 in Italy, before the first COVID-19 cases were described in China. Rapid tests have multiple benefits; however, a confirmation assay is required to avoid false positive results.

Lack of association between Vitamin D status and free light chains profile with different chronic HCV-related liver and extrahepatic disorders.

OBJECTIVE: A still uncertain association between vitamin D levels and HCV chronic liver diseases has been reported. Increased levels of serum-free light chains (FLCs) and an altered k/λ FLC ratio correlate with Mixed Cryoglobulinemia (MC) vasculitis and/or B-cell non-Hodgkin's lymphoma in HCV-positive patients. We aimed to investigate the possible role of vitamin D, vitamin D Binding Protein (DBP), and FLCs levels as a tool for discriminating different stages of HCV- related MC and chronic liver diseases. PATIENTS AND METHODS: Sixty-five untreated patients were retrospectively enrolled and 21 healthy blood donors (HBD) were used as controls. Vitamin D, DBP, FLCs, and cryoglobulins levels were measured. Based on cryoglobulins, patients were divided in three subgroups (without cryoglobulins, type II, and type III). RESULTS: We didn't find any significant differences in vitamin D and DBP levels between HCV patients' main groups and HBD. Serum FLCs levels were significantly higher in HCV patients than in HBD. FLCs ratio among patients' subgroups did not reveal differences. CONCLUSIONS: Our results confirm the presence of an increased serum level of FLCs in HCV patients and suggest that nor vitamin D and DBP or FLC levels can be considered reliable biomarkers for discriminating different stages of HCV-associated chronic liver diseases and/or HCV-associated extrahepatic manifestation. We confirm that serological FLCs levels are significantly higher in patients than in HBD as a signature of B cell activation in course of HCV infection.

A novel biomarker score for the screening and management of patients with plasma cell proliferative disorders.

OBJECTIVE: Monoclonal plasma cell proliferative disorders comprise a wide spectrum of diseases associated to clonal B-cell expansion. Serum protein electrophoretic profile (SPEP) and circulating free light chains (FLCs) levels are the mainstay of diseases management. Recently, soluble (s) Syndecan-1 (SDC1, CD138) produced by myeloma plasma cells has been suggested in the monitoring and follow-up of patients with myeloma. The aim of our study is to evaluate sCD138 in addition with FLCs and SPEP for the screening of patients with different evolutive disease pathways. PATIENTS AND METHODS: Sera from 73 patients with monoclonal gammopathy of undetermined significance (MGUS), 120 smoldering and 42 multiple myeloma (SMM and MM, respectively), 70 HCV-related mixed cryoglobulinemia (MC), 35 B-cell non-Hodgkin's lymphoma (B-NHL) and sera from 50 healthy donors (HD), were tested for sCD138, FLCs (assessed by means of ELISA and turbidimetric assay, respectively) and electrophoresis pattern (performed on Capillarys system) for the generation of a novel biomarker score (BS). RESULTS: Our results were grouped according to the two main lines of disease progression (vs. MM or B-NHL): in one group we found BS mean values of 0.2, 3.4, 5.3, 7.1 for HD, MGUS, SMM and MM, respectively; in the other group of 0.2, 4.4, 6.7 for HD, MC and B-NHL. CONCLUSIONS: We showed that BS mean values follow the ingravescence disease status towards the two main lines of progression to cancerous conditions; it could represent an additional useful tool in the management of screening and/or follow-up.

IgG cryoglobulinemia.

OBJECTIVE: Mixed Cryoglobulinemia is the most well-known Hepatitis C Virus (HCV)-associated extrahepatic manifestation. MC is both an autoimmune and B-lymphoproliferative disorder. Cryoglobulins (CGs) are classified into three groups according to immunoglobulin (Ig) composition: type I is composed of one isotype or Ig class. Type II and type III mixed CGs are immune complexes composed of polyclonal IgGs acting as autoantigens and mono, polyclonal or oligoclonal IgM with rheumatoid factor activity. IgG1 and IgG3 are the predominant subclasses involved. This study shows the simultaneous presence of IgG-RF and IgG3, supporting the hypothesis of an involvement of this subclass in the initiation of early stages of CGs. PATIENTS AND METHODS: We describe a case series of six HCV-positive patients, all of whom had peripheral neuropathy and transient ischemic attacks, presenting cryoprecipitates formed by IgG3 and IgG1. Cryoprecipitate IgG subclass research was carried out by immunofixation electrophoresis by using antisera against IgG1, IgG2, IgG3, and IgG4. RESULTS: Our six patients presented with an immunochemical pattern characterized by the mere presence of IgG1 and IgG3 subclasses with probable RF activity and one of these six patients exhibited monoclonal IgG3 in his cerebrospinal fluid. CONCLUSIONS: We can hypothesize that the IgG passage through the blood-brain barrier could have contributed to the cause of TIAs, through a mechanism involving the precipitation of circulating immune complexes formed by the two subclasses in the intrathecal vessels.

Paradoxical inhibition of cardiac lipid peroxidation in cancer patients treated with doxorubicin. Pharmacologic and molecular reappraisal of anthracycline cardiotoxicity.

Anticancer therapy with doxorubicin (DOX) and other quinone anthracyclines is limited by severe cardiotoxicity, reportedly because semiquinone metabolites delocalize Fe(II) from ferritin and generate hydrogen peroxide, thereby promoting hydroxyl radical formation and lipid peroxidation. Cardioprotective interventions with antioxidants or chelators have nevertheless produced conflicting results. To investigate the role and mechanism(s) of cardiac lipid peroxidation in a clinical setting, we measured lipid conjugated dienes (CD) and hydroperoxides in blood plasma samples from the coronary sinus and femoral artery of nine cancer patients undergoing intravenous treatments with DOX. Before treatment, CD were unexpectedly higher in coronary sinus than in femoral artery (342 +/- 131 vs 112 +/- 44 nmol/ml, mean +/- SD; P < 0.01), showing that cardiac tissues were spontaneously involved in lipid peroxidation. This was not observed in ten patients undergoing cardiac catheterization for the diagnosis of arrhythmias or valvular dysfunctions, indicating that myocardial lipid peroxidation was specifically increased by the presence of cancer. The infusion of a standard dose of 60 mg DOX/m(2) rapidly ( approximately 5 min) abolished the difference in CD levels between coronary sinus and femoral artery (134 +/- 95 vs 112 +/- 37 nmol/ml); moreover, dose fractionation studies showed that cardiac release of CD and hydroperoxides decreased by approximately 80% in response to the infusion of as little as 13 mg DOX/m(2). Thus, DOX appeared to inhibit cardiac lipid peroxidation in a rather potent manner. Corollary in vitro experiments were performed using myocardial biopsies from patients undergoing aortocoronary bypass grafting. These experiments suggested that the spontaneous exacerbation of lipid peroxidation probably involved preexisting Fe(II) complexes, which could not be sequestered adequately by cardiac isoferritins and became redox inactive when hydrogen peroxide was included to simulate DOX metabolism and hydroxyl radical formation. Collectively, these in vitro and in vivo studies provide novel evidence for a possible inhibition of cardiac lipid peroxidation in DOX-treated patients. Other processes might therefore contribute to the cardiotoxicity of DOX.

Increased levels of IGF-1 and beta2-microglobulin in epithelial lining fluid of preterm newborns developing chronic lung disease. effects of rhG-CSF.

UNLABELLED: Insulin-like growth factor-1 (IGF-1) is involved in regulating the Th-1/Th-2 balance, favoring the development of the Th-2 compartment which enhances fibrosis, one of the main characteristics of Chronic Lung Disease (CLD) in premature newborns. Limited data is available concerning a possible association between early epithelial lining fluid (ELF) concentrations of IGF-1 (total and free forms), IGF-binding protein-3 (IGFBP-3), beta2-microglobulin and subsequent development of CLD in preterm neonates. If neutropenic, preterm neonates are frequently treated with recombinant human granulocyte colony stimulating factor (rhG-CSF). The objective of the study was to correlate ELF concentrations of IGF-1 and beta2 microglobulin during the first week of life both in non-neutropenic and in rhGCSF-treated neutropenic preterm neonates, with subsequent development in CLD. Thirty preterm neonates with Respiratory Distress Syndrome (6 with neutropenia) were studied. Eleven out of 24 non-neutropenic preterm infants (46%) and all of the six neutropenic subjects (100%) developed CLD. With the exception of first day values, there was a clear similarity in the behaviors of assayed molecules between non-neutropenic and neutropenic patients developing CLD. Non-neutropenic patients without CLD showed significantly lower values of free IGF-1 and beta2M both on days 1 and 3. Total IGF-I and cell counts were different only on the 3rd day. CONCLUSIONS: 1) the mechanisms leading to CLD might be mediated by high levels of IGF-family molecules soon after birth 2) beta2M could be a marker of increased bronchoalveolar lavage fluid cellularity with potential inflammatory properties 3) G-CSF treatment induces an increased synthesis of IGF-1 molecules by cells recruited in the lung, with possible enhancement of the fibrogenic mechanisms.

Enzyme inactivation by metal-catalyzed oxidation of coenzyme Q1.

Ubiquinol-1 in aerated aqueous solution inactivates several enzymes--alanine aminotransferase, alkaline phosphatase, Na+/K(+)-ATPase, creatine kinase and glutamine synthetase--but not isocitrate dehydrogenase and malate dehydrogenase. Ubiquinone-1 and/or H2O2 do not affect the activity of alkaline phosphatase and glutamine synthetase chosen as model enzymes. Dioxygen and transition metal ions, even if in trace amounts, are essential for the enzyme inactivation, which indeed does not occur under argon atmosphere or in the presence of metal chelators. Supplementation with redox-active metal ions (Fe3+ or Cu2+), moreover, potentiates alkaline phosphatase inactivation. Since catalase and peroxidase protect while superoxide dismutase does not, hydrogen peroxide rather than superoxide anion seems to be involved in the inactivation mechanism through which oxygen active species (hydroxyl radical or any other equivalent species) are produced via a modified Haber-Weiss cycle, triggered by metal-catalyzed oxidation of ubiquinol-1. The lack of efficiency of radical scavengers and the almost complete protection afforded by enzyme substrates and metal cofactors indicate a 'site-specific' radical attack as responsible for the oxidative damage.

Defective plasma antioxidant defenses and enhanced susceptibility to lipid peroxidation in uncomplicated IDDM.

Oxidative stress is postulated to be increased in patients with IDDM. Accumulating evidence suggests that oxidative cell injury caused by free radicals contributes to the development of IDDM complications. On the other side, a decreased efficiency of antioxidant defenses (both enzymatic and nonenzymatic) seems to correlate with the severity of pathological tissue changes in IDDM. Thus, we determined plasma antioxidant defenses, measuring the total radical-trapping antioxidant capacity (TRAP) and the two markers of oxidative stress, lipid hydroperoxides (ROOHs) and conjugated dienes, in 72 patients with well-controlled IDDM and without evident complications, compared with 45 nondiabetic subjects. Compared with control subjects, IDDM patients showed significantly reduced plasma TRAP (669 +/- 131 vs. 955 +/- 104 micromol/l, P < 0.001) and significantly increased levels of ROOHs (7.13 +/- 2.11 vs. 2.10 +/- 0.71 micromol/l, P < 0.001) and conjugated dienes (0.0368 +/- 0.0027 vs. 0.0328 +/- 0.0023 arbitrary units [AU], P < 0.01), especially in the trans-trans conformation (0.0340 +/- 0.0028 vs. 0.0259 +/- 0.0022 AU, P < 0.001), with a concurrent reduction of conjugated dienes in the cis-trans conformation (0.0028 +/- 0.0011 vs. 0.0069 +/- 0.0012 AU, P < 0.001). The oxidative parameters studied did not appear to be correlated with metabolic control (HbA1c levels) and lipid profile (cholesterol or triglyceride levels). The reduced TRAP and the increased ROOH and conjugated diene plasma levels, together with the decreased ratio of cis-trans/trans-trans conjugated dienes, which reflects an altered redox status of plasma, indicate that in IDDM patients, oxidative stress is enhanced and antioxidant defenses are defective, regardless of diabetes duration, metabolic control, or presence of complications.

Large, sustained cardiac lipid peroxidation and reduced antioxidant capacity in the coronary circulation after brief episodes of myocardial ischemia.

OBJECTIVES: We sought to investigate whether a brief episode of myocardial ischemia produces a detectable cardiac oxidative stress in patients undergoing elective coronary angioplasty (PTCA). BACKGROUND: Although cardiac oxidative stress has been clearly demonstrated in experimental models of ischemia-reperfusion, its presence in patients after transient myocardial ischemia is still unclear. METHODS: In order to evaluate oxidative stress in ischemic cardiac regions, plasma conjugated dienes (CD), lipid hydroperoxides (ROOHs) and total antioxidant capacity (TRAP), independent indexes of oxidative stress, were measured in the aorta and great cardiac vein (GCV) before (t0), 1, (t1), 5 (t5) and 15 min (t15) after first balloon inflation in 15 patients undergoing PTCA on left anterior descending coronary artery (Group 1); six patients with right coronary artery stenosis (Group 2), which is not drained by the GCV, were studied as controls. RESULTS: In Group 1 at baseline, CD and ROOHs levels were higher in GCV than in aorta (p < 0.01 for both), and TRAP levels were lower (p < 0.01). Aortic levels of CD, ROOHs and TRAP did not change at any time after to; venous levels of CD and ROOHs levels markedly increased at t1, at t5 and remained elevated at t15 (p < 0.01 for all comparisons vs. to); venous levels of TRAP decreased at t1 and t5 (p < 0.01 vs. t0) and returned to normal at t15. In Group 2, CD, ROOHs and TRAP levels were similar in the aorta and GCV and did not change throughout the study. CONCLUSIONS: Short episodes of myocardial ischemia during PTCA induce a sustained oxidative stress, which is detectable in the venous effluent of reperfused myocardium.

Free radical production by activated haem proteins: protective effect of coenzyme Q.

The interaction of hydrogen peroxide with haem proteins leads readily to the formation of myoglobin and/or haemoglobin higher oxidation states (MbIV and/or HbIV), which are capable of promoting the oxidation of cellular costituents and are probably to blame for myocardic tissue damage in ischaemia/reperfusion. This study supports the evidence that the reduced form of Coenzyme Q, like other reducing agents, has an antioxidant activity exerted through the progressive reduction of ferryl forms (MbIV and/or HbIV) back to met and oxy forms (Mb and/or HbIIO2). Furthermore, the strong inactivation afforded by ferryl states of myoglobin on several enzymes, especially creatine kinase (CK), can be prevented by the addition of ubiquinol which protects the enzyme from the oxidative modifications. The ability of ubiquinol to recycle ferryl states of haem proteins provides a novel antioxidant mechanism for Coenzyme Q, besides its direct or indirect antiperoxidative activity, and may represent an important defense mechanism against oxidative tissue injury.

NA+/K+ ATPase impairment and experimental glycation: the role of glucose autoxidation.

Non enzymatic glycation could be involved in the early impairment of Na+/K+ ATPase that occurs in sciatic nerve of diabetic rats. In fact, decrease of Na+/K+ ATPase activity is one of the first alterations showed in experimental diabetic neuropathy. In this respect, it is known that in the presence of transition metals under physiological conditions, glucose can autoxidize yielding hydrogen peroxide (H2O2) and free radical intermediates, which, in turn, inhibit the cation pump. Our experiments were designed to determine if glucose autoxidation has any relevance in the early steps of Na+/K+ ATPase experimental glycation. Compared experiments with and without the sodium borohydride (NaBH4) reduction step demonstrated that incubation of brain Na+/K+ ATPase with glucose 6-phosphate (G 6-P) and trace metals induced a significant decrease in enzyme activity dramatically enhanced by addition of copper (Cu2+). A concomitant production of H2O2 was noticed. The presence of diethylenetriaminepentaacetic acid (DTPA), a strong metal chelator, completely prevented Na+/K+ ATPase impairment and hydrogen-peroxide formation. No gross structural and conformational alterations of the enzyme can be demonstrated by intrinsic and extrinsic fluorescence measurements. Our results suggest that during the exposure of brain NA+/K+ ATPase to glucose 6-phosphate in vitro (experimental glycation), the decrease in activity can be correlated, at lease in the early phases, to metal-catalyzed production of oxidative species, such as H2O2, through the glucose autoxidation process, and not to glucose attachment to the enzyme. Since plasma hydroperoxides and copper appear to be elevated in diabetic patients with complications, our data suggest a critical role for oxidative reactions in the pathophysiology of the chronic complications of diabetes like neuropathy.

Liver, pancreas and biliary tract enhanced lipoperoxidation products in pure pancreatic juice: evidence for organ-specific oxidative stress in chronic pancreatitis.

BACKGROUND: Oxygen-free radicalscan play a role in the development of chronic pancreatitis, altering the redox state with damage of cell constituents and decrease in antioxidant defences. AIMS: To measure levels of lipoperoxidation products, conjugated dienes and lipid hydroperoxides, in pure pancreatic juice and serum of chronic pancreatitis patients and compare them to that in controls. To investigate a possible correlation with serum indexes of pancreatic inflammation (amylase and lipase). PATIENTS: Pancreatic juice was collected during ERCP, after secretin stimulation, in 20 patients with chronic pancreatitis and 11 controls with biliary diseases. METHODS: Lipid hydroperoxide levels were determined with FOX2 method and measured as absorbance at 560 nm. Conjugated diene levels were measured using second-derivative spectroscopy. RESULTS: No substantial difference was present in serum levels of lipid hydroperoxides, conjugated dienes (in both isomeric forms) and isomer-ratio values between those of patients with chronic pancreatitis and controls. In pancreatic juice, there was a significant increase in lipid hydroperoxides and conjugated dienes levels (especially trans-trans isomers) in chronic pancreatitis patients compared with controls, with a decrease in cis-trans isomers and a significant difference in isomer-ratio values. CONCLUSIONS: Increased levels of lipid hydroperoxides and conjugated dienes in the pancreatic juice of chronic pancreatitis patients is indicative of an enhanced lipoperoxidation and antioxidants consumption in pancreatic tissue, confirmed by the decreased isomer-ratio values as an indirect index of decreased antioxidant capacity. The lack of significant difference in conjugated diene and lipid hydroperoxide levels in the serum of chronic pancreatitis patients versus that of controls suggests an oxidative stress limited to pancreatic tissue and indicative of an organ-specific pathology, confirmed by the parallel behaviour of oxidative parameters (lipid hydroperoxides and conjugated dienes) and indexes of pancreatic inflammation (amylase and lipase).

Oxidative stress in ischemia-reperfusion injury: assessment by three independent biochemical markers.

BACKGROUND: Oxidative stress plays a key role in ischemia-reperfusion injury, causing peroxidation of tissue lipids and proteins. However, it is debated whether brief ischemic episodes are sufficient to cause detectable oxidative stress in humans, since biochemical markers used so far in the setting of percutaneous transluminal coronary angioplasty (PTCA) gave conflicting results. METHODS: We determined lipid hydroperoxides (ROOHs), conjugated dienes (CD) and total radical-trapping antioxidant capacity (TRAP), three different independent markers of oxidative stress, in aortic and great cardiac vein blood of 5 patients undergoing PTCA before a single balloon inflation lasting 115 +/- 38 s (t0), and 1 min (t1), 5 min (t5), 15 min (t15) after balloon deflation (Group 1). ROOHs and CD were also determined in aortic and great cardiac vein blood of 5 patients with mitral valve disease (Group 2). RESULTS: In Group 1, great cardiac vein levels of ROOHs and CD at t1 increased by 219% and 79%, respectively, compared to t0 (p < 0.01); this sharp and consistent increase persisted up to t15 (+189% and +63%, respectively, compared to t0; p < 0.01). Great cardiac vein levels of TRAP were significantly lower than aortic levels at t0, and exhibited a further decrease at tl. No significant differences in aortic and great cardiac vein levels of ROOHs and CD at t0 were observed between Group 1 and Group 2. CONCLUSIONS: The three methods we used showed a remarkable sensitivity for the detection of post-ischemic reperfusion injury in cardiac venous blood and may be useful for detecting small foci of ischemia-reperfusion injury in microvascular angina.

Conformational stability of bovine alpha-crystallin. Evidence for a destabilizing effect of ascorbate.

Short-term incubation of bovine alpha-crystallin with ascorbate alters the protein conformational stability. The denaturation curves with urea and guanidinium-chloride show different patterns, suggesting a deviation from a two-state mechanism owing to the presence of one or more intermediates in the unfolding of ascorbate-modified alpha-crystallin. Furthermore, the latter protein profiles are shifted to lower denaturant concentrations indicating a destabilizing action of ascorbate, which is capable of facilitating protein dissociation into subunits as demonstrated by gel filtration with 1.5 M-urea. The decrease in conformational stability cannot be ascribed to any major structural alteration, but rather to localized changes in the protein molecule. In fact, no difference between native and ascorbate-treated alpha-crystallin can be detected by amino acid analysis but perturbation of the tryptophan and tyrosine environment is indicated by alterations in intrinsic fluorescence. Furthermore, turbidity and light-scattering measurements suggest an involvement of the lysine side chains, since aggregability patterns with acetylsalicylic acid are significantly altered. The ascorbate-destabilizing effect on the conformational stability of alpha-crystallin, probably exerted through oxidative modification of amino acid residues and/or the formation of covalent adducts, provokes unfavourable steric interactions between residues along the polypeptide chains, thus favouring aggregation and insolubilization of crystallins which can lead to cataract formation, as also demonstrated by proteolytic digestion patterns which show a lower rate of degradation of the ascorbate-modified alpha-crystallin.

Inhibition of basal insulin secretion induces insulin resistance in normal man: evidence for a tonic effect of insulin on carbohydrate metabolism.

Insulin resistance has been demonstrated both in insulin deficiency and insulin excess in man and in animals. This study was carried out in normal man to evaluate the role of insulinopenia in the pathogenesis of insulin resistance. Insulin suppression was obtained by 4 h somatostatin (SRIF) infusion. Insulin receptors on circulating monocytes were evaluated before and after SRIF infusion; an insulin tolerance test (ITT) was performed after SRIF, saline or SRIF and replacing basal insulin secretion. Insulin binding to circulating monocytes did not change after 4 h insulinopenia (2.19 +/- 0.30 vs. 2.35 +/- 0.80%), while insulin sensitivity appeared decreased after SRIF (KITT = 0.97 +/- 0.13) as compared with saline (KITT = 3.30 +/- 0.42), and this effect was prevented by insulin (KITT = 2.46 +/- 0.38). A relationship was detected between KITT and plasma insulin concentration before ITT (r = 0.85, p less than 0.01), suggesting that insulin deficiency is the main cause of the phenomenon observed. The present data suggest that basal insulin concentration plays an essential role in the control of insulin sensitivity. If insulin binding on monocytes mimics the behavior of major insulin target tissues, it is possible that the impaired insulin action after 4 h of insulin deficiency is related to a post binding effect.

Mixed function oxidation and enzymes: kinetic and structural properties of an oxidatively modified alkaline phosphatase.

No major structural alteration of alkaline phosphatase can be observed in the early stages of enzyme oxidative inactivation by the ascorbate model system. Fluorescence changes of protein-bound 8-anilino-1-naphthalenesulfonic acid suggest, however, that localized modifications take place. Oxidized alkaline phosphatase displays less catalytic efficiency (decrease of Vmax), while retaining the other kinetic properties, including the same affinity for substrates and inhibitors and the same activation energy of the native enzyme. Typical features of the modified protein are a decreased thermal stability and a biphasic heat inactivation profile, which make the oxidized form quite similar to aged enzymes. The lower response to Mg2+ activation indicates that the magnesium binding sites of alkaline phosphatase are probably the targets of the ascorbate system oxidative modifications.