Biomedical subjects

G Curatola

Publications and source records attributed to G Curatola.

At least 19 recordsLinked to original sources

PubMed2004

Effect of genistein against copper-induced lipid peroxidation of human high density lipoproteins (HDL).

Several studies have demonstrated that the isoflavone genistein exerts a protective effect against lipid peroxidation of low density lipoproteins (LDL). Aim of our study was to investigate whether genistein protects high density lipoproteins (HDL), isolated from normolipemic subjects, against Cu(++)-induced lipid peroxidation. Our results demonstrated that genistein exerts an inhibitory effect against Cu(++)-induced lipid peroxidation of HDL, as shown by the lower increase in the levels of conjugated dienes in lipoproteins oxidized after preincubation with different concentrations of genistein (0.5-2.5microM). Moreover the analysis of fluorescence emission spectra of tryptophan (Trp) and Laurdan (6-dodecanoyl-2-dimethyl-aminonaphthalene) demonstrated that genistein prevents the alterations of apoprotein structure and physico-chemical properties, associated with Cu(++)-triggered lipid peroxidation of lipoproteins. The protective effect exerted by genistein against oxidative damage of lipoproteins was realized at concentrations similar to those observed in plasma of human subjects consuming a traditional soy diet or receiving a soy supplement. Therefore, we suggested that antioxidant activity exerted by genistein against lipid peroxidation of HDL in vitro could be of physiological relevance.

2-Naphthylamine↗

PubMed2004

Chemical-physical properties of lipoproteins in anorexia nervosa.

BACKGROUND: Anorexia nervosa (AN), a psychosomatic disorder, has serious negative effects on multiple organs and systems of the human body. Anorexia nervosa usually runs a chronic course and is associated with significant morbidity and mortality. In order to elucidate the role played by lipids in AN, in the present study we compared the plasma lipid profile and the chemical-physical properties of lipoproteins obtained from subjects affected by AN. MATERIALS AND METHODS: The study was performed on lipoproteins of AN subjects and of age-matched healthy subjects used as controls. We tested the susceptibility to oxidative stress in vitro, the fatty acid content, the fluidity using 2-dimethylamino-(6-lauroyl)-naphthalene (Laurdan) and 1,6-difenil-1,3,5-esatriene (DPH) probes. RESULTS: Present results indicate that AN patients present a deep alteration of the composition and of chemical-physical properties in circulating lipoproteins, even in the absence of significant modifications to clinical metabolic parameters. A significantly decreased body mass index (BMI) was found in AN patients in comparison with controls. Anorexia nervosa patients showed a significant modification of phospholipids to protein ratio and a significantly increased percentage of unsaturated fatty acids compared with control subjects as well as a decreased fluidity, a significantly increased percentage of liquid-crystalline phase in VLDL, and a significantly reduced susceptibility to oxidative stress, more pronounced in LDL. CONCLUSIONS: These results confirm the hypothesis that anorexia is accompanied by changes of lipid metabolism in the central nervous system (CNS).

Adult↗

PubMed2004

Protective effect of paraoxonase activity in high-density lipoproteins against erythrocyte membranes peroxidation: a comparison between healthy subjects and type 1 diabetic patients.

High-density lipoproteins (HDL) plays a key role in the protection against oxidative damage of lipoprotein and biological membranes. The aim of the present study was to investigate the relationship between the antioxidant role of HDL and the HDL-paraoxonase (PON) activity in healthy subjects and in type 1 diabetic patients. Moreover, the ability of HDL of controls and diabetic patients to protect and/or repair biological membranes from oxidative damage was studied. HDL were isolated from 31 type 1 diabetic patients and 31 sex- and age-matched healthy subjects and immediately used to evaluate lipid hydroperoxides and HDL-PON activity. Erythrocyte membranes obtained from healthy subjects were oxidized with 2,2-azo-bis(2-aminidinopropane)dihydrochloride and then incubated in the presence of HDL isolated from healthy or type 1 diabetic subjects, with measurements of membrane lipid hydroperoxides before and after the incubation. HDL from type 1 diabetic patients showed higher levels of lipid hydroperoxides and a lower activity of HDL-PON than healthy subjects. Moreover, HDL of type 1 diabetic patients protected less efficiently erythrocyte membranes against oxidative damage compared with HDL from healthy subjects. A negative correlation was found between HDL-PON activity and the levels of hydroperoxides of HDL, confirming the relationship between PON and lipid peroxidation and suggesting that subjects with low PON activity are more exposed to oxidative damage than subjects with high PON activity. The ability of HDL to protect erythrocyte membranes was positively correlated with HDL-PON activity and negatively correlated with the levels of lipid hydroperoxides of HDL of healthy subjects. These results confirm a linkage between PON activity and lipid peroxidation of lipoproteins and suggest that the ability of HDL to protect erythrocyte membranes might be related to the PON activity. It might be hypothesized that the decrease of PON activity in diabetic patients and the lower HDL protective action against membrane peroxidation could contribute to acceleration of arteriosclerosis in type 1 diabetes mellitus.

Adult↗

PubMed2004

Modification induced by homocysteine and low-density lipoprotein on human aortic endothelial cells: an in vitro study.

The aim of the present study was to investigate the effect exerted by low-density lipoprotein (LDL) modified by homocysteine (Hcy)-thiolactone (Hcy-LDL) on functional properties on human endothelial cells. Hcy-thiolactone, a reactive product formed in human cells from enzymatic conversion of Hcy, was hypothesized to play an important role in Hcy-induced vascular damages. Using endothelial cultured cells [human aortic endothelial cells (HAEC)] as cellular model, we evaluated nitric oxide (NO) production, cytoplasmic Ca(2+) levels, Na(+)/K(+)-ATPase activity, and peroxynitrite production in cells incubated in the presence of control LDL or Hcy-LDL. Homocysteinylation of LDL was carried out by incubation of LDL, isolated from plasma of healthy subjects, with 100 microm Hcy-thiolactone. A significant increase in cytoplasmic Ca(2+) levels and peroxynitrite production and a decrease in Na(+)/K(+)-ATPase and NO production in HAEC incubated with Hcy-LDL compared with HAEC incubated with control LDL were observed. Moreover, a positive correlation was found between Na(+)/K(+)-ATPase activity and cytoplasmic Ca(2+) content and between peroxynitrite activity and cytoplasmic Ca(2+) content. In conclusion, our results demonstrated that LDL homocysteinylated in vitro induced alterations of functional properties and NO metabolism of human endothelial cells.

Adult↗

PubMed2003

Effect of homocysteinylation on human high-density lipoproteins: a correlation with paraoxonase activity.

We investigated the effect of homocysteine (Hcy)-thiolactone on the activity of the enzyme paraoxonase (PON) associated with human high-density lipoprotein (HDL-PON). HDL were isolated from plasma of normolipidemic subjects. The increase in the levels of sulfhydryl groups (-SH) in HDL incubated with Hcy-thiolactone demonstrates that homocysteinylation of HDL occurs. The increase of -SH groups correlated with the basal values of HDL-PON activity (r = -0.73, P <.001, and r = -0.70, P <.002 using 10 micromol/L and 1 mmol/L Hcy-thiolactone, respectively) suggesting a relationship between the susceptibility of HDL to homocysteinylation and HDL-PON activity. A decrease in the activity of the enzyme HDL-PON was observed in homocysteinylated HDL (Hcy-HDL). The negative correlation established between the basal levels of HDL-PON activity and the percentage decrease of HDL-PON activity (r = -0.76, P <.001, and r = -0.86, P <.001 using 10 micromol/L or 1 mmol/L Hcy-thiolactone, respectively) suggests that subjects with higher HDL-PON activity have a lower decrease in PON activity with respect to subjects with lower HDL-PON activity. The positive correlation established between the percentage decrease of PON activity and the percentage increase of -SH groups in Hcy-HDL (r = 0.80, P <.001, and r = 0.76, P <.001 in HDL incubated in the presence of 10 micromol/L and 1 mmol/L Hcy-thiolactone, respectively) suggests that the modifications of HDL-PON activity are likely related to the compositional changes at the lipoprotein surface of Hcy-HDL. The enzyme PON contributes to the protective role of HDL against the oxidative damage and against toxicity exerted by Hcy involved in the development of atherosclerosis. Therefore the significant decrease of the enzyme activity in HDL incubated with Hcy-thiolactone suggests that homocysteinylation could render HDL less protective against oxidative damage and against toxicity of Hcy-thiolactone.

Adult↗

PubMed2002

Effect of human Apo AIV against lipid peroxidation of very low density lipoproteins.

Recent studies have demonstrated that Apo AIV exerts a protective effect against atherosclerosis. Moreover, Qin et al. (Am. J. Physiol. 274 (1998) H1836) have demonstrated that Apo AIV, isolated from rat plasma, exerts an inhibitory effect against Cu(2+)-induced lipid peroxidation of intestinal lymph and LDL. The aim of the study was to investigate whether human Apo AIV exerts a protective effect against Cu(2+)-induced lipid peroxidation. Our results demonstrated that human Apo AIV exerted an inhibitory effect against Cu(2+) and AAPH induced lipid peroxidation of VLDL, as shown by the lower increase in the levels of TBARS and conjugated dienes in lipoproteins preincubated with Apo AIV. In addition, the tryptophan (Trp) and probe 2-(dimethylamino)-6-lauroylnaphthalene (Laurdan) fluorescence studies demonstrated that the modifications of spectral properties in both lipoproteins preincubated with Apo AIV were lower with respect to ox-lipoproteins, suggesting that Apo AIV prevents the modification of physico-chemical properties due to peroxidation.

2-Naphthylamine↗

PubMed2002

Glycated low density lipoproteins modify platelet properties: a compositional and functional study.

The interaction between low density lipoproteins (LDL) and platelets might play a central role in the development of atherosclerosis in diabetes. The aim of the present study was to investigate whether the glycation of LDL is associated with modifications of their physico-chemical and functional properties and to study the action of glycated LDL (glycLDL) on platelets. LDL and platelets were isolated from 15 healthy subjects. The content of thiobarbituric acid-reactive substances and the generalized polarization of the fluorescent probe Laurdan were determined in LDL glycated in vitro. Platelets were incubated with native LDL, GlycLDL, and minimally oxidized LDL, and the following parameters were evaluated: platelet aggregation, nitric oxide production, intracellular Ca(2+) concentrations, Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase), and Ca(2+)-ATPase activities. GlycLDL showed increased thiobarbituric acid-reactive substance levels, a red shift of the Laurdan emission maximum, and a decrease in generalized polarization, indicating a higher polarity and a reduced molecular order compared with native LDL. GlycLDL caused a significant increase in platelet nitric oxide production, intracellular Ca(2+) concentration, and aggregating response to ADP; an inhibition of the platelet membrane Na(+)/K(+)-ATPase activity; and a stimulation of Ca(2+)-ATPase activity. Minimally oxidized LDL did not cause statistically significant changes in the parameters studied. The present work demonstrates that glycation induces compositional and structural changes in LDL and suggests that an altered interaction between glycLDL and platelets might play a role in the vascular complications of diabetes.

2-Naphthylamine↗

PubMed2001

Effect of glycation of high density lipoproteins on their physicochemical properties and on paraoxonase activity.

We investigated the effect of incubation of high density lipoprotein (HDL) under hyperglycaemic conditions on lipid composition, physicochemical properties and activity of paraoxonase (PON), a calcium-dependent enzyme associated with HDL that contributes to the antiatherogenicity of this lipoprotein. HDL incubated for three days with various glucose concentrations (0-100 mM) had significant increases in thiobarbituric acid-reactive substances (TBARS) and conjugated dienes with respect to control HDL. These results suggest that lipid peroxidation accompanies HDL glycation in vitro. The susceptibility to lipid peroxidation was higher in HDL isolated from subjects with low HDL-paraxonase activity with respect to subjects with higher HDL-PON activity. The lipid compositional changes were associated with modifications of apoprotein conformation as shown by the red-shifted position of the maximum emission of tryptophan in treated HDL. The decrease in the Gp (generalized polarization) value and the red-shifted position of the maximum emission of Laurdan incorporated in treated HDL demonstrate modifications of order and polarity with respect to control HDL. The negative correlation established between the Gp value and TBARS demonstrates that the modifications in molecular order are likely related to the increase in lipid peroxidation products. The activity of paraoxonase was significantly decreased in HDL incubated at 37 degrees C; a greater decrease occurred in the presence of 50 mM and 100 mM glucose. This study demonstrates modifications of lipid composition, apoprotein conformation and physicochemical properties of HDL incubated in the presence of glucose. These modifications affect the activity of HDL-associated paraoxonase. The physicochemical properties of lipoproteins play a regulatory role in lipoprotein function. The modification of order and polarity of glycated HDL and the alterations in paraoxonase activity could potentially contribute to the accelerated atherosclerosis in diabetic patients.

2-Naphthylamine↗

PubMed2000

Age-related modifications in human unstimulated whole saliva: a biochemical study.

Human whole saliva contains a number of antimicrobial agents, and lysozyme, lactoferrin, secretory IgA and peroxidase are among the best known. Peroxidase catalyzes a reaction involved in the inhibition of bacterial growth and metabolism, and the prevention of hydrogen peroxide accumulation, thus protecting proteins from the action of oxygen and reactive oxygen species (ROS). To better understand the role played by the oxidative stress in the aging process, we studied the relationship between total protein content, peroxidase activity, malondialdehyde (MDA) and nitric oxide (NO) content of human unstimulated whole saliva in 169 healthy subjects subdivided into groups according to age. Our results show a significant decrease in peroxidase activity with age. Moreover, the increase in saliva lipid peroxide levels indicates an enhanced free radical production that may contribute to tissue damage. On the other hand, findings concerning human unstimulated whole saliva NO content showed a significant increase in elderly subjects, suggesting that an enhanced NO production might depend on a stimulation of leukocyte-inducible NO synthase (i-NOS) activity. Our results suggest that during aging the oral tissues may become more susceptible to environmental factors due to a modification in the balance between different antimicrobial agents.

Adolescent↗

PubMed1999

N-dansyl-S-nitrosohomocysteine a fluorescent probe for intracellular thiols and S-nitrosothiols.

The fluorescence emission spectrum of N-dansyl-S-nitrosohomocysteine was enhanced approximately 8-fold upon removal of the NO group either by photolysis or by transnitrosation with free thiols like glutathione. The fluorescence enhancement was reversible in that it could be quenched in the presence of excess S-nitrosoglutathione. Attempts were then made to utilize N-dansyl-S-nitrosohomocysteine as an intracellular probe of thiols/S-nitrosothiols. Fluorescence microscopy of fibroblasts in culture indicated that intracellular N-dansyl-S-nitrosohomocysteine levels reached a maximum within 5 min. N-Dansyl-S-nitrosohomocysteine fluorescence was directly proportional to intracellular GSH levels, directly determined with HPLC. N-Dansyl-S-nitrosohomocysteine preloaded cells were also sensitive to S-nitrosoglutathione uptake as the intracellular fluorescence decreased as a function of time upon exposure to extracellular S-nitrosoglutathione.

Animals↗

PubMed1999

Physicochemical and functional modifications induced by obesity on human erythrocyte membranes.

BACKGROUND: Na+,K(+)-ATPase activity was evaluated in relation to membrane composition and molecular organization in erythrocyte membranes from obese patients by the amphyphylic molecule 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan). Its possible relationship with fat distribution and hyperinsulinaemia was also investigated. DESIGN: Subjects were 10 obese men (OM), 12 women with subcutaneous obesity (FSO), 10 women with abdominal obesity (FAO) and 41 healthy lean subjects, 26 women (FC) and 15 men (MC). An oral glucose tolerance test was administered to all subjects to evaluate insulin secretion and glucose tolerance. RESULTS: Na+,K(+)-ATPase activity was increased in all obese patients. Values were higher in FSO and FAO than in FC (with FAO greater than FSO) and in OM than in MC. The erythrocyte membrane cholesterol-to-phospholipid ratio was increased in obese patients and was significantly different in FSO patients compared with FC. The erythrocyte membrane protein-to-phospholipid ratio was also increased in all obese subjects, reaching statistical significance only in FSO vs. FC. The liquid crystalline phase, as tested by Laurdan generalized polarization (GP), was decreased in obese patients, indicating the presence of greater molecular environmental order; all patients groups showed lower GP values than control subjects, but only FAO reached statistical significance compared with FC. There was no evident correlation between membrane Na+,K(+)-ATPase activity and insulin levels, nor did membrane composition and properties show any evident relationship with insulin levels. CONCLUSION: Both increased Na+,K(+)-ATPase activity and altered fluidity and lipid composition were observed in the erythrocyte membrane of all obese patients. These findings are in line with previous observations by our group and indicate that the changes in Na+,K(+)-ATPase activity observed in obese patients could be related to changes in plasma membrane organization and composition.

Adult↗

PubMed1999

Influence of low density lipoprotein from insulin-dependent diabetic patients on platelet functions.

In the present work we studied in vitro the action of low density lipoproteins (LDL) isolated from normolipemic insulin-dependent diabetic (IDDM) patients on transmembrane cation transport, nitric oxide synthase (NOS) activity, and aggregating response to stimuli of platelets from healthy subjects to elucidate whether the modified interaction between circulating lipoproteins and cells might be one of the pathogenetic mechanisms of the increased platelet activation in IDDM. LDL were obtained by discontinuous gradient ultracentrifugation from 15 IDDM out-patients and 15 sex- and age-matched healthy subjects and used for incubation experiments with control platelets. Lipid composition and hydroperoxide concentrations were studied in LDL. Platelet aggregation responses to ADP, NOS activity, cytosolic Ca2+ concentrations, and platelet membrane Na+/K+-adenosine triphosphatase (Na+/K+-ATPase) and Ca2+-ATPase activities were measured after incubation. IDDM LDL showed an increased lysophosphatidylcholine content compared with that of control LDL. IDDM LDL significantly increased the platelet aggregating response to ADP, cytosolic Ca2+ concentrations, and plasma membrane Ca2+-ATPase activity and significantly reduced NOS activity and platelet membrane Na+/K+-ATPase activity compared with those of platelets incubated in buffer or cells incubated with control LDL. The effects exerted by IDDM LDL on platelet suspensions from healthy subjects mimic the alterations observed in platelets from diabetic subjects in basal conditions. Both the decreased activity of NOS and the higher cytoplasmic concentrations of Ca2+ might cause increased platelet activation, as observed in IDDM. In conclusion, the present study suggests a new mechanism with a potential role in the early development of atherosclerosis in diabetic patients, i.e. an altered interaction between circulating lipoproteins and platelets.

Adenosine Diphosphate↗

PubMed1999

Changes in the composition of human unstimulated whole saliva with age.

The aim of this study was to evaluate changes in the concentration of certain components of human unstimulated whole saliva during aging, in order to better understand the role played by aging in oral health. In particular, we studied total protein concentration, alpha-amylase activity, sialic acid content and calcium and phosphorus concentrations in 100 healthy subjects of both genders, aged between 10 and 80 years, who were subdivided into four groups according to their age: 10-25 years, 26-40 years, 41-65 years, and 66-80 years. Other than sialic acid, the concentrations of the components studied were not affected by age. There was a significant negative correlation between sialic acid content and age. Our data indicate the presence of a decreased submandibular/sublingual function with aging, thus suggesting the possibility of a concomitant reduction in the modulating action of unstimulated whole saliva on the oral flora.

Adolescent↗

PubMed1999

Study of fluidity of low density lipoproteins from liver cirrhotic patients.

BACKGROUND AND AIM: Liver disease is accompanied by major quantitative and qualitative modifications in plasma lipoprotein metabolism. Alterations in plasma lipoprotein composition and a lower susceptibility to in vitro peroxidation of low density lipoprotein (LDL) and erythrocyte membranes have been observed in liver cirrhosis. The main objective of the present work was to investigate LDL chemical composition and fluidity in liver cirrhosis using the fluorescence polarization (Pf) of the 1,6-diphenyl-1,3,5-hexatriene (DPH) probe. METHODS AND RESULTS: The chemical composition of LDL was studied in 12 cirrhotic patients and 22 controls by conventional methods and its fatty acid composition by gas chromatography. LDL fluidity was determined by measuring the DPH Pf values. A decrease in molecular order was demonstrated by the significant (p < 0.05) decrease in Pf values in the cirrhotics. Modifications in LDL fluidity are correlated with its composition. A significant increase in triglyceride content (p < 0.05), and significant increases in triglyceride/protein and triglyceride/phospholipid ratios were observed in the cirrhotics. CONCLUSIONS: Our results demonstrate that the higher LDL fluidity of cirrhotic patients may be due to an increased triglyceride content.

Case-Control Studies↗

PubMed1998

Physico-chemical properties of copper-oxidized very low density lipoproteins.

The aim of our study was to investigate the effect of Cu2+ catalyzed oxidation on VLDL physico-chemical properties and secondary structure of apo B-100. Incubation of very low density lipoproteins with copper ions resulted in a decrease in tryptophan and lysine residues parallel to lipid peroxidation products, conjugated dienes and TBARS. Fluorescence polarization showed an increase in the molecular order at the lipoprotein surface of VLDL, as demonstrated by the increase in Pf values of DPH. The secondary structure of apo B-100 was investigated by infrared spectroscopy. Increased order and structural changes, as observed after oxidative stress on VLDL, could be of relevance in the abnormal interactions between lipoproteins and cell membranes.

Apolipoproteins B↗

PubMed1998

Decreased nitric oxide synthase activity in platelets from IDDM and NIDDM patients.

Nitric oxide (NO) produced by platelet nitric oxide synthase (NOS) inhibits platelet activation by increased cytoplasmic cGMP levels. The aim of this study was to investigate platelet NOS activity in insulin-dependent (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM), which are characterized by enhanced platelet activation. HbA1c levels, platelet NOS and platelet membrane Na+/K+ ATPase activity were determined in 19 IDDM patients, 21 NIDDM patients and 31 healthy control subjects. NOS activity was measured by a spectrophotometric method based on NO-dependent oxidation of oxyhaemoglobin to met-haemoglobin. Na+/K+ ATPase activity was measured by the method of Kitao and Hattori. Both NOS and Na+/K+ ATPase activity were significantly reduced in diabetic subjects compared with control subjects. NOS showed a significant negative relation with HbA1c levels and a positive relation with Na+/K+ ATPase activity in diabetic patients. It is hypothesized that the decreased NOS activity might play a role in the pathogenesis of diabetic vascular complications.

Adult↗

PubMed1997

Reduced beta-strand content in apoprotein B-100 in smaller and denser low-density lipoprotein subclasses as probed by Fourier-transform infrared spectroscopy.

The secondary structure of apolipoprotein B-100 in low-density lipoprotein (LDL) subfractions was analysed by Fourier-transform IR spectroscopy. LDLs were isolated in three density ranges by gradient centrifugation of human plasma from healthy volunteers. The spectra revealed differences in the lipid content and composition of the three LDL fractions. The secondary structure of apolipoprotein B-100 was the same in the two fractions corresponding to the large less-dense LDL particles, whereas a lower content of beta-strands was found in the third fraction corresponding to the smaller denser LDL particles. Analysis of the spectroscopic data suggests that, in the same set of LDL subfractions, the particle size is probably the cause of the observed differences in apolipoprotein B-100 secondary structure.

Apolipoprotein B-100↗

PubMed1997

Sialic acid, diabetes, and aging: a study on the erythrocyte membrane.

Sialic acid (SA) content and Na+/K+-ATPase activity of red blood cell (RBC) membranes were studied in 26 normoalbuminuric patients with insulin-dependent diabetes mellitus (IDDM), 25 normoalbuminuric patients with non-insulin-dependent diabetes mellitus (NIDDM), and 40 healthy nondiabetic subjects with a negative family history for diabetes. A decrease in RBC membrane SA content and Na+/K+-ATPase activity was observed in older control subjects compared with younger controls. A significant correlation between age, Na+/K+-ATPase activity, and SA content was also found. No difference was observed in RBC membrane SA content between IDDM and NIDDM subjects, but Na+/K+-ATPase activity was significantly lower in IDDM patients. SA content was increased in NIDDM subjects compared with healthy subjects of similar age, whereas Na+/K+-ATPase activity was significantly lower in both IDDM and NIDDM subjects compared with controls. In NIDDM, Na+/K+-ATPase activity was significantly correlated with age, whereas both Na+/K+-ATPase activity and SA content were significantly correlated in IDDM and NIDDM patients. Hemoglobin A1c, (HbA1c) levels did not show any significant correlation either with Na+/K+-ATPase or with SA content in diabetic patients. The modified SA content and Na+/K+-ATPase activity in elderly subjects described in the present study indicate a similar behavior of the erythrocyte membrane during both RBC senescence and aging of subjects.

Adolescent↗