Population pharmacokinetic modeling of sustained release lithium in the serum, erythrocytes and urine of patients with bipolar disorder.

PURPOSE: Lithium (Li), the first-line treatment of bipolar disorder, was first developed as an immediate-release form with a routine therapeutic drug monitoring 12 h after the last dose. In Europe, the most commonly prescribed form is a sustained release (srLi). Yet no pharmacokinetics (PK) study has been published of srLi, administered once a day, in adults. The present study describes srLi PK in the serum and erythrocytes of bipolar patients. METHODS: To assess srLi PK, we studied prospectively 17 French bipolar patients on a median dose of 1000 mg (600-1600) for at least 2 years. Serum (S), erythrocyte (E) concentrations, and urinary (U) amount were collected over 8 h after 15 days of morning intake using monitoring electronic medical system (MEMs). Population PK parameters were estimated using the SAEM algorithm (MONOLIX 4.3.3 software). RESULTS: Using a population approach, we built a PK population model of srLi including one S compartment (VS = 23.0 L, ClS = 1.21 L h-1), one E compartment (VE = 64.7 L, ClSE = 3.63 L h-1, ClES = 9.46 L h-1), and one U compartment (F = 0.62) and estimate the ratio of concentrations to Li in E over S at 0.38 with 27% between-subject variability. CONCLUSION: This is a PK model of srLi once a day in bipolar patients using a population approach simultaneously describing Li concentrations in serum, erythrocytes, and urine which provide an estimate of the ratio of concentration in erythrocyte over serum and its between-subject variability (BSV).

Exchangeable copper: a reflection of the neurological severity in Wilson's disease.

BACKGROUND AND PURPOSE: The severity of Wilson's disease (WD) is linked to free copper accumulating in the liver and brain. Exchangeable copper (CuEXC) is a new technique to determine plasmatic copper and is useful in the diagnosis of WD. It is hypothesized that it may also enable a good evaluation of extra-hepatic involvement and its severity. METHODS: Forty-eight newly diagnosed WD patients were prospectively evaluated using hepatic, neurological, ophthalmological and brain magnetic resonance imaging (MRI) scores. Three phenotypic presentations were distinguished: pre-symptomatic, hepatic and extra-hepatic. CuEXC was determined in addition to standard copper assays before decoppering therapy. Correlations between biological parameters and the different scores were determined and compared in the hepatic and extra-hepatic groups. RESULTS: Extra-hepatic patients had significantly higher CuEXC values than those with the hepatic form (P < 0.0001). The overall ability of CuEXC to separate the two forms was satisfactory, with an area under the curve of 0.883 (95% confidence interval 0.771-0.996) and an optimal threshold for extra-hepatic diagnosis of 2.08 µmol/l (sensitivity 85.7%; specificity 94.1%). In extra-hepatic patients, CuEXC was the only biological marker to be positively correlated with the Unified Wilson Disease Rating Score (r = 0.45, P = 0.016), the Kayser-Fleischer ring score (r = 0.46, P = 0.014) and the brain MRI score (r = 0.38, P = 0.048), but it was not correlated with the hepatic score. CONCLUSIONS: Exchangeable copper determination is useful when diagnosing WD as a value >2.08 µmol/l is indicative of the severity of the extra-hepatic involvement. In the case of purely hepatic presentation, atypical or mild neurological signs, it should encourage physicians to search for lesions in the brain and eyes.

Usefulness of lead isotopes analysis in a pediatric case of chronic lead poisoning.

Identifying the source of lead is a key measure in the management of childhood lead poisoning. In cases with multiple potential sources or with unusual circumstances, this identification can be complex. By comparing the lead isotopic ratios (IRs) between the potential sources and the blood sample from the patient, it is possible to identify which source caused the poisoning. We report here the case of a 13-years-old girl, screened for lead poisoning as she was known to eat paint chips from two walls in her apartment. Her blood lead level was 395 µg/L, and the environmental investigation came back negative. X-ray fluorescence found lead concentration in paints to be below the regulatory threshold of 1 mg/cm2. As there was no other potential source of lead exposure in this case, a comparative analysis of the IRs of lead between the child's blood and two presumed sources (bathroom and kitchen paints) was performed. This analysis confirmed the source of lead intoxication to be the paints in the apartment, mostly from the bathroom wall.

Corpus callosum abnormalities in Wilson's disease.

INTRODUCTION: Wilson's disease (WD) with neurological presentation is associated with brain lesions classically localised in globus pallidus, putamen, thalamus, mesencephalon, pons and dentate nucleus. Lesions of corpus callosum (CC) have not been studied in a broad population of patients with WD. OBJECTIVE: Evaluation of the frequency of CC lesions in patients with neurological symptoms related to WD. METHOD: The authors included all patients with neurological expression of WD, followed in the French national centre for WD who had a brain MRI between March 2006 and December 2008. The localisation of brain lesions was analysed and the frequency of lesions in CC evaluated. All patients were assessed using the Unified Wilson's Disease Rating Scale. For patients with abnormalities located in CC, a clinical dysconnexion syndrome was investigated. RESULTS: Among 81 patients (45 men, mean age: 34.8 years, from 12 to 74 years) with neurological expression, 42% had white-matter lesions on fluid-attenuated inversion recovery MRI. 23.4% of patients presented CC lesions, limited to the posterior part (splenium). The severity of disability estimated by Unified Wilson's Disease Rating Scale was correlated with the presence of CC lesions on MRI. CONCLUSION: Abnormalities in CC are not unusual (23.4%). Together with lesions of basal ganglia, CC signal changes should suggest the diagnosis of WD.

[An unrecognized cause of myelopathy associated with copper deficiency: the use of denture cream]. / Une cause méconnue de myélopathie par carence en cuivre: l'utilisation de pâte adhésive dentaire.

We report two patients with myelopathy associated with copper deficiency and pancytopenia. Excessive intake of zinc can lead to a severe deficiency of copper reducing the absorption of ingested copper. The patients had in common consumption of denture adhesive paste containing zinc. In both patients, laboratory tests showed a combination of copper deficiency, hyperzincemia and increased urinary zinc level. The use of a denture cream was stopped. Copper supplementation, initially subcutaneously then oral corrected the copper deficiency and pancytopenia. Clinically, the pain faded but the gait disturbance persisted. Copper deficiency associated with the use of denture cream rich in zinc is an unrecognized cause of myelopathy associated with pancytopenia which should be diagnosed early to establish appropriate therapeutic measures to minimize neurological complications.

Identifying sources of lead exposure for children, with lead concentrations and isotope ratios.

Despite a dramatic decrease in children's blood lead levels (BLL), lead exposure remains a public health concern because increasing evidence shows effects at very low doses. Lowering BLL still further requires the identification of lead sources and, therefore, new tools to investigate and thus prevent exposure. We describe a procedure that uses both lead concentrations and isotope ratios (IRs) to identify sources of overexposure in homes. Water, dust, and paint chips were sampled from the homes of 21 children with elevated BLL from Aubervilliers (Paris metropolitan area). Lead concentrations of concern were calculated from reverse physiologically based pharmacokinetic modeling for water and dust. Isotope ratio matching of blood and environmental samples (with a lead content above the concentration of concern) was performed by computation of the distance between their IRs. When the IR of the source did not match that of the blood, the source was eliminated as a source of lead intoxication. The number of sources eliminated (per child) due to lead concentration ranged from 14% to 86% (mean 66%) for dust, and 100% for water samples. The number of remaining potential sources eliminated by IR interpretation varied from 0% to 100% for both dust and paint chips (mean 63% and 58%, respectively). IRs made it possible to eliminate at least one source in 20 of 21 cases and identified a single source in 11 of 21. The number of dust and paint sources not eliminated by concentration or IR varied from 8% to 45% (median 18%). The pilot study supports the usefulness of these procedures and the added value of IRs for identifying sources of lead poisoning. However, systematic use should be supported by cost-effectiveness analysis on a larger and more representative population of elevated BLL.

[Serum selenium and dilated cardiomyopathy in Cotonou, Benin]. / Sélénium plasmatique et cardiomyopathie dilatée à Cotonou (Bénin).

PURPOSE: Peripartum cardiac failure is common in savannah-Sahelian Africa. It is due to a form of dilated cardiomyopathy (DCM) known as peripartum cardiomyopathy (PPCM) that results from undetermined causes. Numerous risk factors have been identified and dietary selenium deficiency has been proposed as a possibility. The purpose of this study was to measure serum selenium levels in patients presenting cardiac insufficiency due to PPCM and DCM (nonpostpartum) in Cotonou, Benin and to compare patients with healthy postpartum women with comparable or identical obstetric features. METHODS: Measurements of selenium status were performed in 10 women (mean age, 27.1 years) with PPCM, 18 patients of both sexes (male/female, 11/7; mean age, 38.9 years) with DCM (non-peripartum), and 46 healthy recent post partum women (mean age, 29.8 years). The Wilcoxon nonparametric test was used for data analysis with a statistical significance level at a p-value <0.05. RESULTS: Mean serum selenium values were near similar in patients presenting PPCM and DCM (non peripartum): 94 +/- 12 microg/L versus 97 +/- 19 microg/L respectively. This difference was not significant. No value was less than 72 microg/L. Conversely significantly lower mean values were observed in the 46 healthy recent postpartum women: 76 +/- 13 microg/L (p = 0.0002). The lowest value was 57 microg/L. CONCLUSION: Serum selenium measurements in Cotonou showed that levels were higher in patients presenting PPCM or DCM (nonperipartum) than in healthy controls. Extremely low values were never observed. These results differed from those obtained in Bamako, Mali and in Niamey, Niger and argue against considering selenium deficiency as a risk factor for development of DCM (peripartum or not) in Cotonou.

[Pineapple juice as a negative oral contrast agent in magnetic resonance cholangiopancreatography]. / Utilisation du jus d'ananas en tant qu'agent de contraste négatif en cholangiopancréatographie par résonance magnétique*

PURPOSE: The quality of magnetic resonance cholangiopancreatography (MRCP) images is frequently degraded by high signal from the gastrointestinal tract on heavily T2W images. The purpose of this study is to evaluate pineapple juice (PJ) as an oral negative contrast agent in MRCP. MATERIALS AND METHODS: Results from MRCP in 50 patients with PJ and 50 patients with paramagnetic contrast (ferumoxsil-Lumirem) were compared. Reviewers were blinded to the type of contrast agent. Exam quality was recorded with regards to signal suppression in the stomach, duodenum and proximal small bowel and with regards to pancreatic duct and biliary ducts visualization. In vitro, the signal characteristics of several commercially available brands of PJ were assessed using T1W, T2W and MRCP sequences. Signal intensity was correlated with the manganese concentration measured using atomic absorption spectrometry. Finally, the reviewers compared the taste of PJ and ferumoxsil. RESULTS: On MRCP sequences, results were similar with regards to signal suppression in the stomach, duodenum and proximal small bowel with PJ and ferumoxsil. Visualization of the pancreatic duct, intrahgepatic bile ducts and CBD was similar with PJ and ferumoxsil. The signal intensity of commercially available brands of PJ on T2W and MRCP sequences correlated well with the measured manganese concentration on spectroscopy. Variations in manganese concentration were observed, with values ranging from 3.65 to 27.24 mg/L. The reviewers noted that PJ tasted "good" or "very good" and that ferumoxsil tasted "bad" or "very bad". CONCLUSION: Ingestion of PJ provides effective signal suppression in the GI tract on MRCP, similar to paramagnetic contrast agents. Because manganese concentration is highly variable in commercially available PJ brands, a brand with high manganese concentration should be selected.

Ethanol oxidation into acetaldehyde by 16 recombinant human cytochrome P450 isoforms: role of CYP2C isoforms in human liver microsomes.

The involvement of cytochromes P450 (CYPs) in the oxidation of ethanol into acetaldehyde was investigated by using 16 recombinant human CYP isoforms. Apparent K(m) and V(m) were determined for CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9*1, CYP2C9*2, CYP2C9*3, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP4A11. All of the tested CYPs, except CYP2A6 and CYP2C18, metabolized ethanol into significant amounts of acetaldehyde and displayed K(m) values around 10mM. The significant correlation found between ethanol oxidation and CYP2E1, CYP3A4 and CYP1A2 catalytic activities in a panel of human liver microsomes confirmed the strong implication of these CYPs in ethanol metabolism. The contribution of CYP2C isoforms which are the most abundant in the liver after CYP3A4, was studied using selective inhibitors either with recombinant CYP2C isoforms or in human liver microsomes. Tienilic acid (100 microM) and ticlopidine (20 microM), mechanism-based inhibitors of CYP2C9 and CYP2C19, respectively, decreased ethanol oxidation by 8+/-1.2% and 7.6+/-1.6% in human liver microsomal samples while selective inhibitors of CYP2E1 (DEDTC 100 microM), CYP3A4 (TAO 50 microM) and CYP1A2 (furafylline 25 microM) decreased it by 11.9+/-2.1%, 19.8+/-1.9% and 16.3+/-3.9%, respectively. As ethanol can be metabolized by most of CYPs, it helps to explain or predict alcohol-xenobiotics interactions which are of high importance in medical prescription.

The embalming of John of Lancaster, first Duke of Bedford (1435 AD): A forensic analysis.

During the Middle Ages and Renaissance, embalming the cadaver of the elite was common practice, being a highly technical treatment mixing vegetal and mineral substances. To assess the exact kind of embalming reserved for the dead body (with the practical necessities of desiccation and good odour), we performed a full biomedical analysis of the mummified remains of John Plantagenet of Lancaster, first Duke of Bedford, regent of France for his nephew, the English King Henri VI (died 1435 AD). Here, we show, among other aspects, that the body was embalmed using substances whose origins were in apothecary and botany: mercury, myrtle, mint, frankincense, lime and, possibly, cinnamon and copper.

A clinical and pharmacological study of arsenic trioxide in advanced multiple myeloma patients.

We previously showed that arsenic trioxide (ATO) and melarsoprol may inhibit the growth of multiple myeloma (MM) cells in vitro and in vivo. We report here the administration of arsenic derivatives in 12 relapsing or refractory secretory MM patients. A total of 10 patients received ATO (eight in a continuous schedule, two discontinuously) and two received melarsoprol. The melarsoprol arm was prematurely closed due to toxicity. In the ATO arm, median duration of treatment was 38 days (9-54). Hepatic toxicity was grade 3 and 2 in one and eight patients, respectively. Other toxicities included neuropathy (n=2, grade 2), encephalitis (n=1, grade 3) and leuconeutropenia (n=4, grade 3). At 2 weeks after treatment initiation, mean serum concentration of arsenic was 1.11+/-0.16 micromol/l. No complete or partial remission was observed. A minor response (25-49% reduction of M protein in serum) and a stabilization of the M-protein level were observed in three and four patients, respectively. After ATO discontinuation, these responses were of short duration in all cases. ATO as a single agent did not produce any significant response in advanced MM patients despite sufficient arsenic exposure. Strategies to improve biodistribution, pharmacokinetic and efficacy of the drug as well as treatment combinations are needed.

Lipoprotein(a) in diabetic patients with and without chronic renal failure.

OBJECTIVE: To examine the distribution of Lp(a) plasma levels in patients with IDDM and NIDDM, and in nondiabetic and IDDM patients with chronic renal failure. RESEARCH DESIGN AND METHODS: Cross-sectional study of Lp(a) plasma levels in a population of diabetic patients with stable metabolic control, with simultaneous determination of plasma lipids, fasting plasma glucose, and HbA1. Thirty-six patients with IDDM, 90 with NIDDM, and 41 with chronic renal failure (20 IDDM, 21 nondiabetic) were compared with 78 control subjects. RESULTS: Lp(a) plasma levels were significantly higher in IDDM and NIDDM patients, as well as in nondiabetic and IDDM patients with chronic renal failure compared with control subjects. No correlation was observed between Lp(a) and lipid plasma levels, fasting plasma glucose, and HbA1. CONCLUSIONS: Lp(a) may contribute to the increased prevalence of atherosclerotic disease in diabetic patients and patients with chronic renal failure, especially in IDDM patients whose lipoprotein pattern was not different from that of the control group.

Lithium chloride antileukemic activity in acute promyelocytic leukemia is GSK-3 and MEK/ERK dependent.

We recently identified that the MEK/ERK1/2 pathway synergized with retinoic acid (RA) to restore both transcriptional activity and RA-induced differentiation in RA-resistant acute promyelocytic leukemia (APL) cells. To target the MEK/ERK pathway, we identified glycogen synthase kinase-3ß (GSK-3ß) inhibitors including lithium chloride (LiCl) as activators of this pathway in APL cells. Using NB4 (RA-sensitive) and UF-1 (RA-resistant) APL cell lines, we observed that LiCl as well as synthetic GSK-3ß inhibitors decreased proliferation, induced apoptosis and restored, in RA-resistant cells, the expression of RA target genes and the RA-induced differentiation. Inhibition of the MEK/ERK1/2 pathway abolished these effects. These results were corroborated in primary APL patient cells and translated in vivo using an APL preclinical mouse model in which LiCl given alone was as efficient as RA in increasing survival of leukemic mice compared with untreated mice. When LiCl was combined with RA, we observed a significant survival advantage compared with mice treated by RA alone. In this work, we demonstrate that LiCl, a well-tolerated agent in humans, has antileukemic activity in APL and that it has the potential to restore RA-induced transcriptional activation and differentiation in RA-resistant APL cells in an MEK/ERK-dependent manner.

Effect of fat-emulsion phospholipids on serum lipoprotein profile during 1 mo of cyclic total parenteral nutrition.

Changes in serum lipoprotein determined by selective precipitation were investigated in 11 adult patients during 1 mo of parenteral nutrition. Patients were divided into two groups that received a similar nutrient regimen except for Intralipid (IL) phospholipid, which was higher in group A (10% IL, n = 5) than in group B (20% IL, n = 6), 139 +/- 15 vs 71 +/- 0.5 mg.kg-1.d-1 (P less than 0.01). Lipoprotein X (LPX) detected soon after IL infusions were started reached its highest concentrations in group A. LPX concentrations correlated with phospholipid intakes on days 7 and 15 but not on day 29. Significant increases in the cholesterol and phospholipid content of low-density-lipoprotein-very-low-density-lipoprotein fractions were observed only in group A. It is suggested that these changes were induced by the twofold-higher intake of phospholipids in group A. With regard to the possible involvement of LPX in lipid overloading of the reticuloendothelial system and hepatocytes, administration of 20% IL seems preferable to 10% IL.

A sequential and simple determination of zinc, copper and aluminium in blood samples by inductively coupled plasma atomic emission spectrometry.

A sequential method of measuring zinc, copper and aluminium in serum by inductively coupled plasma (ICP) spectrometry is described. It involves a 1/5 dilution of serum with a potassium chloride solution which enhances aluminium signal intensity and reduces variations between different matrix compositions. The method is as sensitive as atomic absorption for zinc (sensitivity: 0.11 mumol/l) and copper (sensitivity: 0.020 mumol/l) and can also be applied to monitor aluminium (sensitivity: 0.12 mumol/l) for patients receiving total nutrition therapy or hemodialysis. Its linearity extends at least to 200 mumol/l for copper and zinc and to 20 mumol/l for aluminium. The correlations with atomic absorption are satisfactory for the 3 parameters, as assessed by the correlation coefficients established for both methods. A reference interval was established with 34 sera of control subjects (19 men, 15 women) which showed an average zinc, copper and aluminium of 14.5 (S.D. 2.6), 17.3 (S.D. 2.1) and 0.32 (S.D. 0.12) mumol/l, respectively. This method does not require a simultaneous ICP spectrometer and can be performed with 1 ml of serum in a single tube, using a routine sequential ICP spectrometer.

Lead accumulation in the mouse ovary after treatment-induced follicular atresia.

Although the main target of lead (Pb) toxicity is the red blood cell, Pb-associated changes in the nervous system, the kidney, and the reproductive system have also been described. The few Pb studies conducted on females revealed mostly miscarriages, premature delivery, and infant mortality in humans and animals. This study was done to correlate Pb accumulation in the ovary with damage to folliculogenesis. Pb burden was assayed by atomic absorption spectrometry in bone, liver, adrenal glands, ovary, and fetuses taken from mice exposed according to 2 protocols: intraperitoneal (i.p.) injection of Pb(NO(3))(2) 10 mg/kg/day for 15 days or 10 mg/kg/week for 15 weeks. Ovaries were examined histologically. Pb accumulation in the various soft tissues of acutely exposed mice was similar, and significantly higher than in the organs of chronically exposed mice. A low Pb concentration in the ovary caused dysfunction of folliculogenesis, with fewer primordial follicles and an increase in atretic antral follicles.

Dynamic study about metal release from titanium miniplates in maxillofacial surgery.

It has been shown that titanium (Ti) has had dramatic success in many surgical procedures as a result of its excellent mechanical properties and resistance to corrosion. There is still concern, however, about the release of metal and controversy surrounding whether or not the plates should be removed after bone healing. This study has been conducted to investigate whether or not there is a relationship between duration of plating and metal release from Ti miniplates in maxillofacial surgery. A prospective cohort study design was used. The concentration of Ti, in the soft tissues covering the plates, was examined in all patients who underwent removal of Ti miniplates from January 1998 to April 1999 (51 cases). Inductively Coupled Plasma-Optical Emission Spectrometry was used to measure Ti. Total ti and soluble Ti levels were compared to duration of plating (ranged from 15 days to 3 years, mean = 8 months). Correlation coefficients and two-way ANOVA were data processed. The average amount of total Ti in the soft tissues surrounding the plates was 1306 micrograms/g dry tissue. The mean of soluble Ti was 0.53 microgram/g dry tissue. The results of this study do not support the existence of a relationship between duration of plating and total Ti (correlation coefficient = 0.093 (P > 0.1) nor soluble Ti (correlation coefficient = 0.009 (P > 0.1) in the soft tissue surrounding the plates. Moreover, the only independent factor of Ti release found was associated with mechanical constraints during surgery. Almost 100% of Ti is released during the osteosynthesis. Then Ti levels remain constant in the surrounding tissues. Most of the time, Ti seems to be clinically inert. Compared to the possible risks of a second operation, removal of Ti miniplates should not be a routine procedure except in the case of complaints from patients, particularly in the case of infection, hypersensitivity, dehiscence or screw loosening.

Physiological chromium determination in serum by Zeeman graphite furnace atomic absorption spectrometry. A serious challenge.

Several authors have already underlined chromium implication in glucose and lipids metabolism of humans. In this field, physiological chromium determination in serum could be helpful, but the discrepancies reported in numerous papers are confusing. Here we report some results obtained by Zeeman correction Graphite Furnace Atomic Absorption Spectrometry. This technique includes a dilution of serum with 12.5 mM ultrapure nitric acid and 0.25% Triton X-100 (final concentrations). Some main features can be outlined: (1) the contamination constitutes a serious drawback and (2) the sensitivity of the technique is critical (characteristic mass found: 1.76 pg/0.0044 A.s). Our results obtained from 27 healthy subjects (2.01 +/- 0.77 nmol/L) agree with most recent studies and indicate that serum chromium level does not seem to be sex-related.

[Value and limits of determining serum cobalt levels in patients with metal on metal articulating prostheses]. / Intérêt et limite du dosage du cobalt sérique chez les patients porteurs d'une prothèse à couple métal-métal.

PURPOSE OF THE STUDY: The purpose of this study was to measure the serum cobalt levels and their correlation with clinical and radiological findings in patients with metal on metal hip articulating surfaces. METHOD: Forty-one patients with metal on metal hip arthroplasty were reviewed retrospectively at mean follow-up of 12.9 months. Serum cobalt levels were determined for each patient by atomic absorption spectrometry at the maximal follow-up and were compared to a control group (19 patients). Two patients and one control subjects also performed exercise on a treadmill in order to appreciate the influence of physical activity on serum cobalt levels. RESULTS: The metal on metal group presented higher serum cobalt levels than those of the control group (p < 0.0001). There was no correlation between serum cobalt and clinical and radiological findings at the exception of patient age (n = 40, r = 0.37). However, when the follow-up was greater than 18 months, mean serum cobalt was significantly higher compared to a follow-up less than 18 months. The physical exercise test led to a moderate elevation (around 10 p. 100) of cobalt in the two patients but not in the control subject. DISCUSSION AND CONCLUSION: The interpretation of an elevated cobalt serum level is difficult. Cobalt-containing drugs, other implants, excess of activity and diseases (renal failure) may influence serum cobalt level. In this study, the high serum cobalt levels seem not linked to a failure of the implant, mainly because of the short follow-up. They could rather be attributed to an increase of the patient activity beginning 18 months after the surgery. Because potential long-term cobalt toxicity and carcinogenicity is not well known, careful medical follow-up should be emphasized specially in young patients.