Evaluation of Body Composition and Nutritional Status in Patients with Inborn Errors of Metabolism

TESE DE DOUTORAMENTO Evaluation of Body Composition and Nutritional Status in Patients with Inborn Errors of Metabolism Nisreen Abdelaziz Salem Aldmour ESCOLA DE DOUTORAMENTO INTERNACIONAL PROGRAMA DE DOUTORAMENTO EN INVESTIGACIÓN CLÍNICA EN MEDICINA SANTIAGO DE COMPOSTELA 2020 3 Declartion of conflicts of interests: The doctoral candidate declares no conflicts of interest related to her thesis. 5 DECLARACIÓN DO AUTOR DA TESE Evaluation of Body Composition and Nutritional Status in Patients with Inborn Errors of Metabolism Dña. Nisreen Abdelaziz Aldmour Presento a miña tese, seguindo o procedemento axeitado ao Regulamento, e declaro que: 1) A tese abarca os resultados da elaboración do meu traballo. 2) De selo caso, na tese faise referencia ás colaboracións que tivo este traballo. 3) A tese é a versión definitiva presentada para a súa defensa e coincide coa versión enviada en formato electrónico. 4) Confirmo que a tese non incorre en ningún tipo de plaxio doutros autores nin de traballos presentados por min para a obtención doutros títulos. En Santiago de Compostela, ... de ..... de 2020 Asdo: Nisreen Abdelaziz Aldmour AUTORIZACIÓN DOS DIRECTORES DA TESE: Evaluation of Body Composition and Nutritional Status in Patients with Inborn Errors of Metabolism Dña. María Luz Couce Pico Dña. María Rosaura Leis Trabazo INFORMAN: Que a presente tese, correspondese co traballo realizado por Dña. NISREEN ABDELAZIZ ALDMOUR, baixo a nosa dirección, e a utorizamos a súa presentación, considerando que cumpre cos r equisitos exixidos no R egulamento de Estudos de Doutoramento da USC, e que como directores desta non incorre nas causas de abstención establecidas na Lei 40/2015. En Santiago de Compostela, ... de ..... de 2020 Asdo. María Luz Couce Pico Asdo. María Rosaura Leis Trabazo AUTORIZACIÓN DO TITOR DA TESE: Evaluation of Body Composition and Nutritional Status in Patients with Inborn Errors of Metabolism Dña. María Rosaura Leis Trabazo INFORMAN: Que a presente tese, correspondese co traballo realizado por Dña. NISREEN ABDELAZIZ ALDMOUR, baixo a nosa dirección, e a utorizamos a súa presentación, considerando que cumpre cos r equisitos exixidos no R egulamento de Estudos de Doutoramento da USC, e que como directores desta non incorre nas causas de abstención establecidas na Lei 40/2015. En Santiago de Compostela, ... de ..... de 2020 Asdo. María Rosaura Leis Trabazo viii SUMMARY Inborn errors of metabolism (IEM) are genetically based entities generally characterized by enzyme deficiencies that result in the accumulation of toxic compounds in the body. Children with IEM must follow a special diet restricted in essential nutrients, which can put them at risk for nutritional disorders. This study's objective is to evaluate body composition and nutritional status in patients with IEM and controls, as well as their relationship to lifestyles, food intake, and physical activity. Ninety-nine patients with IEM between the ages of 5 and 19 years and 98 controls by age and sex were analyzed to determine their anthropometric characteristics and body composition using DEXA. Data on personal and family history, socioeconomic status, food intake, and physical activity were collected through validated questionnaires, and nutritional biomarkers were analyzed in all of them. Height z-score was significantly reduced in IEM patients (-0.28 vs. 0.15, p = 0.008), affecting more those with carbohydrate metabolism defects followed by those with amino acid metabolism disorders. Adiposity does not present significant differences in these patients and controls (z-score of the body mass index 0.56 vs. 0.42, p = 0.279). The z-score of waist circumference is higher in patients (-0.08 vs. -0.58, p = 0.005). With regard to bone mineralization, patients present a significantly lower bone mineral density (BMD) (0.89 vs. 1.6, p = 0.001), assuming a higher risk of osteopenia (z-score < -2: 33.3% vs. 20.4%) and osteoporosis (z-score < -2.5: 7.1% vs. 0%), none of which has presented fractures. There was a significant positive correlation between natural protein intake and BMD. Among the analytical studies, the low levels of selenium and high levels of folate stand out, particularly in amino acids metabolism disorders. Our results indicate that patients with IEM in dietary treatment, especially those with disorders of amino acid metabolism, present alterations in body composition, such as low height, a tendency to overweight and obesity, and a decrease in bone mineral density. Keywords: Bone mineral density, dietary intake, genetic disorders, nutritional biomarkers, nutritional risk. x xi ABBREVIATIONS AAF Amino Acid Formula AAs Amino Acids AD Autosomal Dominant AR Autosomal Recessive BF Body Fat BH4 Tetrahydrobiopterin BIA Bioelectric Impedance Analysis BMD Bone Mineral Density BMI Body Mass Index BMT Bone Marrow Transplantation CG Classic Galactosemia CK Creatinine Kinase CPT2 Carnitine Palmitoyltransferase deficiency2 DEXA Dual-energy X-ray absorptiometry EAA Essential Amino Acid ECF Extracellular Fluids FAOD Fatty Acid Oxidation Defects. FFM Fat Free Mass FM Fat Mass. GALT Galactose-1-phosphate uridyltransferase. GSD Glycogen Storage Disorders. HELLP Haemolysis, Elevated Liver Enzymes, Low Platelets. HFI Hereditary Fructose Intolerance. IEIPM Inborn Errors of Intermediary Protein Metabolism. IEM Inborn Errors of Metabolism. LC-FAOD Long Chain Fatty Acid Oxidation Disorders. LCHADD Long chain HydroxyacylCoA Dehydrogenase deficiency LSDs Lysosome storage disorders. MADD Multiple acyl-CoAdehydrogenase deficiency. MBD Mineral bone disease. MCADD Medium chain acyl-CoA dehydrogenase. xii MMA Methylmalonic Acidemia. MPS Mucopolysaccharidoses. MS/MS Tandem Mass Spectrometry MSUD Maple Syrup Urine Disease NBS Newborn Screening. NTBC 2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3 -cyclohexanedione. PA Propionic Acidemia. PAH Phenylalanine Hydroxylase. Phe Phenylalanine. PKU Phenylketonuria. RDI Recommended Dietary Intake. SRT Substrate reduction therapy. SSIEM Society for the Study of Inborn Errors of Metabolism. TBW Total Body Water. UCDs Urea Cycle Defects. UNICEF The United Nations International Children’s Emergency Fund. VLCAD Very long chain acyl-CoA dehydrogenase deficiency. WHO World Health Organization. TABLE OF CONTENTS Acknowledgments ........................................................................ iii Resumo .......................................................................................... v Resumen ...................................................................................... vii Summary....................................................................................... ix Abbreviations ............................................................................... xi 1. INTRODUCTION .................................................................... 1 1.1 Inborn errors of metabolism (IEM) ..................................... 1 1.2 Body Composition Measurements ..................................... 44 2. JUSTIFICATION .................................................................... 55 3. OBJECTIVES .......................................................................... 57 3.1 Main outcomes .................................................................. 57 3.2 Secondary .......................................................................... 57 4. MATERIAL AND METHODS ............................................... 59 4.1 Patients ............................................................................... 59 xiv 4.2 Method ............................................................................... 61 5. RESULTS ................................................................................ 73 5.1 General characteristics of sample....................................... 73 5.2 Anthropometric characteristics .......................................... 81 5.3 Body Composition Assemment.......................................... 92 xv 5.4 Patterns of Physical Activity ........................................... 100 5.5 Dietary Contribution and Correlation with Body Composition. ................................................................................ 104 5.6 Biochemical and Haematological Markers. ..................... 119 6. DISCUSSION ........................................................................ 127 6.1 Anthropometric Characteristics. ...................................... 127 6.2 Body Composition Assessment ....................................... 130 6.3 Physical Activity and Body Composition in IEM Patients…… ……... ..................................................................... 135 6.4 Dietary Intake and Correlation with Body Compositio…………………………………………………..…..136 6.5 Biochemical and Haematological Markers ...................... 140 7. CONCLUSIONS ................................................................... 145 xvi Bibliography .............................................................................. 147 List of Tables ............................................................................. 181 List of Figures ............................................................................ 185 Appendix .................................................................................... 187 Appendix A ............................................................................ 187 Appendix B ............................................................................ 191 Appendix C ............................................................................ 207 INTRODUCTION 1 1. INTRODUCTION 1.1 Inborn errors of metabolism (IEM) Definition Metabolism is the regulated and coordinated set of chemical reactions that take place in a living organism; for these chemical reactions to be carried out, the correct functioning of proteins called enzymes is necessary. These enzymes can act at the level of the general metabolism or of a particular cycle, as a cellular receptor, a membrane transporter, or as part of a cellular organelle (Patel et al., 2011). Inborn errors of metabolism (IEM), also known as hereditary metabolic disorders, are genetic alterations with a deficiency or abnormality of an enzyme or its cofactor that is responsible for a clinically significant point in a metabolic pathway. As a result, an abnormal build-up of a substrate or deficiency of the product is recognized. In the majority, this is due to a single gene defect that encodes a particular enzyme important in the metabolic pathway (ElHattab, 2015). IEM are rare diseases from an individual point of view, but, as a whole, they are responsible for significant pediatric morbidity and mortality (Nassogne et al., 2005; McBryde et al., 2006). The European Union (EU) defines rare diseases when there is a risk of death or chronic disability and prevalence of fewer than 5 cases per 10,000 inhabitants. This translates into an estimate of 29 million affected in the EU and 3 million in Spain (Palau, 2009). The first studies on IEM were in 1908 when Archivald E. Garrod, a professor of Medicine at Oxford, coined this term and described four inherited metabolic diseases (cystinuria, alkaptonuria, pentosuria and albinism) (Galton, 2008). In 1934, Ivar Asbjorn Folling described the phenylketonuria (PKU) correlated with mental retardation. Bickel in 1953 published the results of dietary therapy in PKU (Bickel, 1953). INTRODUCTION 8 diversity of presenting symptoms but can be divided into 3 diagnostically useful groups: – Group 1: Disorders of Intermediary Metabolism affecting Small Molecules IEM in this group have plasma and/or urine metabolic marker (i.e., small diffusible water-soluble molecules) that can be easily and rapidly measured. These markers are also useful for therapy monitoring since most of these IEM are responsive to treatment. This group includes IEM that lead to acute or progressive intoxication from the accumulation of normal or unusual compounds proximal to the metabolic block. They encompass classical inborn errors of amino acid catabolism (PKU, MSUD, homocystinuria, tyrosinemia type 1), most organic acidurias (MMA, PA, isovaleric, biotin responsive multiple carboxylase deficiency (MCD), galactose (classic galactosemia), and fructose (hereditary fructose intolerance (HFI)) metabolism defects. Metal disorders can behave in two different ways, like intoxication in cases of accumulation such as in (Wilson disease, hemochromatosis, neuroferritinopathies) and as neurodegeneration with brain iron accumulation syndromes In case of deficiency of small molecules symptoms result primarily from the defective synthesis of compounds that are distal to the block or from the defective transport of an essential molecule through cellular or organelle membranes. Clinical signs are, at least in theory, treatable by providing the missing compound. Most of these defects affect neurodevelopment, have a congenital presentation (antenatal), and may present with birth defects. Inborn errors of neurotransmitter and brain amino acid synthesis are also included in this group, because they share many biochemical characteristics: their diagnosis relies on plasma, urine, and cerebrospinal fluid investigations (amino acid, organic acid analyses, etc); and some are amenable to treatment even when the disorder is present in utero. INTRODUCTION 9 – Group 2: Disorders involving primarily energy metabolism Consist of IEM with symptoms due to a deficiency in energy production or utilization within the liver, myocardium, muscle, brain, and other tissues. Diagnosis can be done by functional tests measuring glucose, lactate, ketones, and other energetic molecules (AA and acylcarnitines) in blood and urine, confirmed by enzyme assays and molecular testings. Membrane carriers of energetic molecules (glucose, lactate, and pyruvate) are the most important molecules involved in energetic carrier defects. These disorders are treatable or partially treatable. Cytoplasmic energy defects are generally less severe. They include glycogen metabolism, gluconeogenesis, hyperinsulinism, which are all treatable, creatine metabolism disorders, which are partially treatable, and pentose phosphate pathways, untreatable with a phenotype mostly linked to defective NADP/NADPH production. Mitochondrial defects are the most severe and are generally untreatable. They encompass aerobic glucose oxidation defects with congenital lactic acidemia (pyruvate transporter, pyruvate carboxylase, pyruvate dehydrogenase system, and Krebs cycle defects), mitochondrial respiratory-chain disorders, mitochondrial transporters of energetic and other indispensable molecules, coenzyme Q biosynthesis, FAO, and ketone body defects. Mitochondrial diseases are clinically diverse and can present at any age. They can manifest in a specific tissue or multisystem manner, most often organs with the highest energy demands such as brain, skeletal muscle, eyes, and heart. - Group 3: Disorders Involving Complex Molecules Include diseases that interrupt the synthesis, processing, and catabolism of complex molecules that take place in mitochondria, lysosomes, peroxisomes, and Golgi apparatus. In this group, clinical symptoms are permanent, very often progressive, and independent of intercurrent events, unrelated to food intake. INTRODUCTION 10 This group includesLSD, peroxisomal disorders, CDG syndrome, inborn errors of purine and pyrimidine, inborn errors of cholesterol and bile acid synthesis, inborn errors of intracellular triglycerides, phospholipids and glycosphingolipids synthesis and remodelling. It also includes glycogen storage depletion, Phospholipids (PL), glycosphingolipids (GSL). Mainly related to the synthesis and recycling of these molecules, which take place in organelles. They may interfere with fetal development. Most present as neurodevelopmental or neurodegenerative disorders unrelated to food intake. Inborn errors of cholesterol and bile acid synthesis present either with multiple malformation syndromes, neonatal cholestasis, or with late onset neurodegenerative disorders. Many other defects affecting systems involved in intracellular vesiculation trafficking, and processing of complex molecules can be anticipated. For example, the CEDNIK neurocutaneous syndrome owing to mutation of SNAP29 implicated in intracellular vesiculation, as well as by mutations in AP5Z1, the cellular phenotype of which bears striking resemblance to features described in a number of lysosomal storage disorders. Due to the great diversity of IEM and their low individual prevalence, determine that both the diagnosis of suspicion and the biochemical-molecular analysis for diagnostic confirmation and treatment are complex. Clinical Presentation of IEM IEM may present at various ages in different ways. Clinical presentation of the disease can occur even before birth, at birth, or during the first days of life as deterioration after normal birth and delivery (Saudubray et al., 1999) or in later stages. Errors in fetal metabolism may be correlated with developing maternal complications most frequent during pregnancy, such as HELLP syndrome (hemolysis, elevated liver enzymes, low platelets), and the fatty liver (Wilcken et al., 2003). At birth, IEM can manifest as INTRODUCTION 11 perinatal asphyxia, or later as nonspecific chronic manifestations such as delays in childhood development. Acute metabolic decompensation in the neonatal age may also present as acidosis, or hyperammonemia (Waters et al., 2018). Most IEM babies born at term seem to be well but then worsen quickly, at the beginning of food intake. Also, in moderate forms, they can manifest symptoms in late childhood, adolescence or even in adulthood. Symptoms usually appear due to changes in catabolism, resulting in an accumulation of metabolites that cause toxic signs. The rate of deterioration is variable according to disease type, the extent of the disease, and what is the affected organ. For example, symptoms and signs of debut may be neurological, unexplained hypoglycemia, cardiomyopathy, hepatic failure, or sudden death. Other diseases have more complex presentations, such as odor that is not generally detected (Leonard and Morris, 2000). In general, IEM can affect more than one system or organ, or they have a localized effect (Colonetti et al., 2018). IEM are responsible for a significant part of childhood disability and deaths (Ferreira et al., 2019). The United Nations International Children’s Emergency Fund (UNICEF), in the statistics of mortality of children mentioned that 9% corresponds to congenital anomalies1. Mainly the signs and symptoms resulting from IEM can be divided into the early-onset and late-onset forms (Vassili and Tien, 2013). Early-Onset Signs and Symptoms Disorders such as non-ketotic hyperglycinemia, multiple deficiency of Acyl-CoA dehydrogenase (MADD), cobalamin defects, and sometimes UCDs are known to begin during the prenatal period (Illsinger and Das, 2010; Mouchegh et al., 2007). 1 https://www.unicef.org/media/47626/file/UN-IGME-Child-Mortality-Rep INTRODUCTION 12 The neonate has a limited repertoire of responses to serious diseases and usually manifests itself with nonspecific symptoms that could be easily attributed to infection or some other common cause. However, there are more common forms of presentation alert symptomatology that help us to guide the diagnosis (Couce et al., 2008; Raghuveer et al., 2006; Burton, 1998). Figure 2. Figure 2: Early onset (Neonatal Period to Infancy) signs and symptom. Neurological distress Most of IEM that result in intoxication or energy deficiency is brought to medical attention because of neurological deterioration. In the case of intoxication, the initial symptom free interval varies in duration depending on the condition. Metabolic encephalopathy: present in a newborn after a few hours or healthy days begins without apparent cause with digestive and neurological symptoms, presenting weak suction, vomiting, lethargy and quickly enters a coma, with changes in muscle tone and involuntary movements (movements of boxing and pedaling in MSUD), and dehydration. There may be abnormal smell of urine and skin. Tests INTRODUCTION 13 routinely performed on sick infants, such as chest x-ray, cerebrospinal fluid examination, bacteriological studies, brain ultrasound are usually normal. The unexpected and the "mysterious" deterioration after a normal initial period is the most important indicator. It can manifest the disorders of protein metabolism (UCDs, organic acidurias, leucinosis). Seizures: are the most distinctive sign of neurological disease in the newborn period and can be caused by a broad range of systemic and central nervous system disorders. Among the metabolic defects, several treatable metabolic disorders can present in the neonatal period or early in infancy predominantly with ‘intractable’ seizures: pyridoxine responsive seizures, pyridoxine-5’-phosphate oxidase deficiency (responsive to pyridoxal phosphate but not to pyridoxine), 3phosphoglycerate dehydrogenase deficiency and other inborn error of serine synthesis (Hart et al., 2007) (responsive to serine supplementation) and persistent hyperinsulinaemic hypoglycaemia. Folinic acid responsive epilepsy is probably not a true entity, but rather corresponds to undiagnosed B6responsive seizures (Gallagher et al., 2009). Biotin responsive holocarboxylase synthetase deficiency can also, albeit rarely, present predominantly with neonatal seizures. GLUT1 deficiency (brain glucose transporter), which can be treated with hyperketotic diet, and biotinidase deficiency can also present in the first months of life as epileptic encephalopathies. Biotin sensitive holocarboxylase synthetase deficiency occurs very rarely with neonatal seizures. There are other recently described disorders that have severe early seizures in which a possible treatment has been suggested (Van Hove and Lohr, 2011; Parisi et al., 2015). Neurological distress with lactic acidosis: infants with lactic acidosis present a diagnostic problem. A high plasma lactate can be secondary to hypoxia, cardiac disease, infection, or convulsions, whereas primary lactic acidosis may be caused by disorders of pyruvate metabolism and respiratory chain defects. Some IEM (FAO, organic acidaemias, and UCD) may also be associated with a secondary lactic acidosis (Poggi-Travert et al., 1996). In a neonate who was not asphyxiated and who has no evidence of other organ failure, the persistent increase of plasma lactate above 3 mmol/L should lead to INTRODUCTION 14 further investigations for an IEM. The most likely causes are abnormalities of pyruvate metabolism (pyruvate carboxylase or pyruvate dehydrogenase deficiency) or a respiratory chain deficit. The presence of cerebral pseudo cysts or myelination abnormalities may suggest a pyruvate carboxylase deficiency. It may also be due to the multiple carboxylase deficiency. Hypo and hypertonia: hypotonia are more generally observed in severe non-metabolic neuromuscular disorders. Metabolic hypotonias are observed in congenital hyperlactacidemias, respiratory chain disorders, UCD, non-ketotic hyperglycinemia, sulphite oxidase deficiency, peroxisomal disorders, Lowe syndrome and tri-functional protein deficiency. Prader-Willi syndrome, one of the most frequent causes of neonatal hypotonia, can simulate hypotonia-cystinuria syndrome. Serious forms of Pompe disease can mimic respiratory chain disorders or tri-functional enzyme deficiency when generalized hypotonia is associated with cardiomyopathy, but it does not start strictly many times in the neonatal period (Saudubray, 2012). Neonatal hypertonia is very common in sulphite oxidase deficiency and in hyperplexia due to abnormal glycerinergic transmission (mutations in receptors and transporters). Severe Ponto cerebellar hypoplasia’s, neonatal forms of Krabbe and gangliosidosis can also produce great hypertonia with signs of hyper excitability. Hepatic and gastrointestinal presentation The involvement distinguished in 6 predominant patterns: • Hepatocellular insufficiency: liver failure with vomiting, jaundice, coagulation disorder, may be due to classic galactosemia, fructosemia (in rare cases if sucrose is administered). Another cause is tyrosinemia type 1, this usually manifests after the 3rd week of life. Transaldolase deficiency and respiratory chain disorders are also possible (Kelly et al., 1993). Severe fetal growth delay, lactic INTRODUCTION 15 acidosis, hyperaminoaciduria, very high concentrations of ferritin, hepatic hemosiderosis and early death alert to Gracile syndrome (Visapä et al., 2002). • Hepatomegaly with hypoglycemia and seizures: this sign give alert to gluconeogenesis defects; type I or III glycogenesis. Severe hyperinsulinism may present with moderate hepatomegaly. • Cholestasis: genetic disorders are an important cause of neonatal cholestasis, and α-1antitrypsin deficiency accounts for a significant portion of these cases. Prolonged neonatal cholestasis jaundice associated with progressive hepatosplenomegaly is the most common sign in NiemannPick disease type C. Spontaneously resolves by 2–4 months of age in most patients, it may lead to liver failure in about 10 % of cases (Brunetti-Pierri et al., 2012). • Hepatosplenomegaly: in the neonatal period is rare but can be seen with signs of storage (coarse faces, macroglossia, fetal hydrops, ascites, oedema, multiple dysostosis, and vacuolated lymphocytes) in lysosomal disorders (GM1 gangliosidosis, sialidosis type II, Niemann-Pick A, MPS type VII, galactosialidosis), and congenital erythropoietic porphyria (Schwarz et al., 2004). • Hepatic steatosis: hepatic presentations of inherited FAO disorders and UCD consist in acute steatosis or Reye syndrome with normal bilirubin rather than true liver failure. Long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency is an exception, which may present early in infancy (but not strictly in the neonatal period) as cholestasis jaundice, liver failure and hepatic fibrosis (Saudubray, 2012). INTRODUCTION 16 • Congenital diarrhea disorders: can be caused by mutations in genes related to disacaridase deficiency, like defect of transport of ions or nutrients by mutations in SLC26A3 causing congenital hydrochloride, pancreatic insufficiency. Affected newborns have vomiting, colic pain and nonbloody watery diarrhea, protein losing enteropathy, hypoalbuminemia, and hyperlipidaemia. Cardiac involvement Some disorders can present predominantly with cardiac disease. Cardiac failure and a dilated hypertrophic cardiomyopathy (pure dilated cardiomyopathy is infrequent), most often correlated with hypotonia, muscle weakness and failure to thrive, suggests FAO, respiratory chain disorders (with severe lactic acidosis), Pompe disease or fatal congenital heart glycogenesis due to mutations in the gene that makes an enzyme called acid α-glucosidase (GAA), (Burwinkel et al., 2005). Antenatal manifestations: malformations, fetal hydrops. Antenatal manifestations can be classified into three main clinical categories (Van Spronsen et al., 2005): • True major malformations: such as skeletal malformations, congenital heart disease, visceral aplasia’s, and neural tube defects. • Dysplasia’s, for example: cortical heterotopias, cortical cysts, polycystickidneys, and liver cysts. • Functional manifestations (intrauterine growth retardation, hydrops fetalis, hepatosplenomegaly and microcephaly). INTRODUCTION 17 According to this classification, irreversible major malformations are only observed in O-glycosylation disorders or secondary to SLC39A8 mutations in the manganese transporter; in defects of cholesterol synthesis; in disorders of amino acid synthesis, such as glutamine and asparagine synthetase deficiency (lissencephaly), and rarely in severe energy defects such as MADD; some respiratory chain disorders; and in the defect of mitochondrial thiamine pyrophosphate carrier SLC25A19 responsible for Amish lethal microcephaly. The defect of the ω−3 fatty acid transporter (MFSD2A) causes significant abnormalities in brain convolutions and premature death. The vast majority of "true intoxication" disorders (amino acids and organic acid catabolism disorders) do not interfere with embryo-fetal development. Highlight of cholesterol biosynthesis defect, S. SmithLemli-Opitz, in which there is a deficiency of 7-dehydrocholesterol reductase, and manifests with microcephaly, cleft palate, cardiac malformations, polydactyly, genitourinary abnormalities and in 2/3 of the cases. Also, in the disorders of the peroxisome biogenesis, rhizomelic chondrodysplasia punctuates with facial malformations and rhizomelic limb shortening, as well as alterations at the respiratory, ocular, skeletal, and physical and mental development levels. Zellweger syndrome or cerebro-hepato-renal syndrome with deep hypotonia, neonatal seizures, difficulty feeding, delayed psychomotor development and usually presents with malformations in the face and skull, liver disorders, with hepatomegaly and hepatic fibrosis, and renal cysts (Saudubray, 2012). Late-Onset Signs and Symptoms Those with higher residual enzyme activity generally present later in life. The presentation may be seen in childhood, adolescence, or even during adulthood. The clinical representation is usually made up of recurrent attacks during the chronic progressive course (Saudubray et al., 2006; Barends et al., 2014). INTRODUCTION 24 Newborn screening (NBS) NBS is applied at presymptomatic identification of specific genetic, metabolic, or infectious states by using tests that can be applied to the entire population of newborns. Screening programs are considered as an essential activity in the context of preventive actions in public health. NBS objective is the early identification and treatment of affected individuals so that timely medical intervention avoids neurological damage, reduces morbidity, mortality, and possible disabilities associated with these diseases. The origin of the NBS is located in the United States, when Guthrie, in the sixties of the last century, launched an analytical procedure for the measurement of Phe in which he used as a biological sample capillary blood obtained from the heel of the newborn and impregnated in filter paper. In 1961 Bickel introduced the Guthrie method to Europe. Helped by Guthrie himself, he set up the first European newborn screening laboratory in Marburg, Germany, which began receiving samples in 1962. Spain was one of the few European countries where Guthrie's influence was noticeable (Couce et al., 2008; Yuan et al., 2015). A widely accepted definition of NBS stated that screening is the application of a test for the identification of individuals at risk of suffering a specific disease to benefit from additional research or treatments among people who have not attempted medical attention because of the symptoms of that disease by (Wald, 1994). NBS is, therefore, the set of actions aimed at the systematic detection of congenital diseases of metabolism in neonatal age (Cocho de Juan et al., 2006). Therefore, NBS has proven effective and efficient, both from diagnosis and from that of public health and economic profitability. An important step in recent years has been the application of tandem mass spectrometry (MS-MS) to the systematic analysis of the dried blood sample collected on the filter paper card to assess metabolism or IEM. This advance allows the detection of most amino acid metabolism INTRODUCTION 25 disorders, organic acidurias, and fatty acid oxidation defects. It is a very sensitive and specific technique, with few false-positive results, which has been incorporated as part of the NBS programs (Dulín-Íñiguez et al., 2006). Tandem mass spectrometry (MS/MS) It is a very useful technique for neonatal screening, late diagnosis, and for disease progression. The development of the technique of MS/MS for the diagnosis of congenital metabolic diseases begins in the 70s, but it is in the 90s when this technology begins to be applied for NBS. The basic methodology for the neonatal screening of metabolic diseases by MS / MS was developed by Millington (Millington et al., 1990). MS/MS is a technique of multiple separation and identification of analysts based on the specific pattern of ionic fragmentation that each compound produces under certain conditions of analysis, and on the separation-detection of each ionic species according to its mass / charge ratio. It is thus possible to separate, detect and quantify in the same test, without the need for an additional chromatographic system and from a single disc of dried blood on filter paper, the amino acids and acylcarnitines that are used as biomarkers, which makes detection possible of congenital metabolic diseases that affect the urea cycle, amino acids, organic acids and β-oxidation of fatty acids with high sensitivity and specificity (Bodamer et al., 2007; Waisbren, 2006).The analysis is very fast (2-3 minutes) and only requires limited sample preparation (Chace et al., 2003). The application of this technology has a very important advance in neonatal screening and has also made it possible to expand biomedical knowledge about different metabolic diseases, in terms of their genetic and phenotypic heterogeneity, since it has allowed us to discover metabolic deficiencies in parents asymptomatic when studying their children (Walter et al., 2009; Wilcken, 2008). INTRODUCTION 26 Mass spectrometry is an analytical technique that provides information on previously analysed molecules converted into ions. The molecules of interest are generally part of a heterogeneous mixture that does not necessarily require a previous separation and are first subjected to an ionization source where they are ionized acquiring negative or positive charge. The ions pass through the mass analyser until they reach different parts of the detector according to their mass / charge ratio (m / z). Once in contact with the detector, signals are generated that are registered in the computer system and represented in a mass spectrum that shows the relative abundance of the signals based on their m / z ratio. Mass spectrometry analysis basically comprises four processes: ►Ionization of the sample. ►Acceleration of ions by an electric field. ►Dispersion of the ions according to their mass / charge. ►Ion detection and production of the corresponding electrical signal. The ionization of the sample can be carried out under different conditions, depending on the nature of the sample itself and what is intended to be detected in the analysis. It can be done in high vacuum conditions, by electronic impact, or at atmospheric pressure. In a liquid phase analysis, an electrospray ionization (ESI) will be performed. In this case, the analyte is introduced into the solution source by an injection pump or from the eluate of a liquid chromatography system. The analyte passes through a stainless steel or silica quartz capillary tube, to which a high potential difference is usually applied in the range of 2.5 to 6 kV. This forces the fogging of the charged drops in the capillary, with a surface charge of the same polarity as the capillary itself. The drops are repelled from the capillary to the cone of the sampling electrode sampling source. With the help of a high temperature in the source of ESI and / or nitrogen gas, as the drops cross the space between the capillary and the cone, they are INTRODUCTION 27 continuously reducing their size, by evaporation of the solvent, which implies an increase of the surface charge density as the radius of the drops decreases. Finally, the force of the electric field in the charged drops reaches a critical point at which the passage to the gas phase for the surface ions is kinetically and energetically possible. At that time, the surface tension is not able to maintain the load (Rayleigh limit), so there is a "Coulombic explosion" and the drop breaks into smaller drops. The process is repeated until only ions remain. Figure 3. Figure 3: Schematic representation of electrospray ionization (ESI) process The main disadvantage presented by this technique is that it produces very little or no fragmentation, so for studies in which a structural assessment is necessary, the use of tandem mass spectrometer is necessary to achieve such fragmentation. In the gas phase, a chemical ionization or a photoionization will be performed. It is a process that uses a large injection flow (0.2-2.0 mL/min) and is applied to non-polar molecules, thermally stable compounds, normally weighing less than 1300 uma. Nebulizer gas and current discharge are required to produce ionization. The process consists mainly of three steps: ion ionozation by nebulizing gas, forming of reactive ions, and reaction with analyte molecules. Figure 4. INTRODUCTION 28 Figure 4: Atmospheric Pressure Chemical Ionization (APCI) . Through a high voltage, the nebulizing gas (air or nitrogen) is ionized forming the first ions. These first ions react immediately with solvent molecules forming reactive ions. Reactive ions react with analyte molecules forming in positive or negative mode. Genetic diagnosis Genetic investigations play a significant role in IEM diagnosis. There are three methods employed for molecular genetic testing: a traditional, well established, and more precise method of Sanger sequencing, and a more advanced method known as Next generation sequencing (NGS). Both traditional and Sanger methods allow for sequencing of genes, determining the sequence of base pairs in the DNA of the exons/exon-intron boundaries or coding regions of a gene. The NGS utilizes the multiplexing of all the sequencing fragments and thus can sequence any number of DNA fragments simultaneously. The NGS genetics has allowed a very considerable advance in the diagnosis of IEM. NGS is now becoming the standard diagnostic methodology in most of the genetic laboratories, including metabolic disorders. (Ghosh., et al., 2017). INTRODUCTION 29 Treatment Significant development has been achieved for the treatment of IEM, especially during the past ten years. Many studies are still being carried for better therapies aiming to cure nearly all IEM. Early diagnosis is essential to initiate early treatment in IEM, which can help to prevent morbidity and mortality. The initial treatment of IEM is aimed to avoid as possible the clinical and biochemical manifestations of the disease and establish irreversible complications while trying to maintain adequate growth and development in the child. See Figure 5. Even today, the treatment poses big problems, since restoring total normality is still an illusion in the IEM (Fernández, 1999). The treatment approaches in IEM can be investigated in two main topics: acute and chronic treatment. Acute treatment includes all the measures to be taken to approach and treat a patient with a severe attack resulting from a suspected IEM (Treat hypoglycemia, hyperammonemia, and acidosis) (Baumgartner et al., 2014). Figure 5: Treatment strategies of IEM disorders. INTRODUCTION 30 According to Ezgu the chronic therapeutic approaches for IEM can be categorized into four groups (Ezgu, 2016): Substrate reduction therapy (SRT) SRT has recently gained broad interest. In addition to directly correcting the enzyme defect, SRT aims to reduce the bioavailability of the compound that cannot be fully metabolized by the defective enzyme (‘substrate reduction’), thereby recovering a steady-state balance of the pathway by lowering the accumulating substrate. Dietary treatment of PKU is often considered the first original application of SRT (Schiffmann, 2015). SRT has gained broad appeal because of the possible administration using small molecule inhibitors that can be taken orally. SRT would particularly apply to the intoxication type of IEM, where toxic metabolite accumulation leads to acute clinical symptoms (Matalonga et al., 2017). Reducing substrate accumulation applied by manipulation of diet, chelation, or biosynthesis inhibition. Gaucher and Niemann-Pick type C diseases are treated by an iminosugar called Miglustat, which inhibits the enzyme glucosylceramide synthase (Cox et al., 2015; Patterson et al., 2007). In some disorders, the abnormal metabolic pathway can be blocked at a different level to prevent toxic substrate accumulation. In tyrosinemia type 1, the deficiency leads to an accumulation of succinylacetone, which is toxic especially for the kidney and the liver. To prevent its accumulation, the pathway is blocked successfully by the inhibitor 2-(2-nitro-4-trifluoromethylbenzoyl)-1.3 cyclohexanedione (NTBC) which can the prevent toxic substrate accumulation (MasurelPaulet et al., 2008). Genistein instead has been shown to reduce the accumulation of glycosaminoglycans in mucopolysaccharidoses (MPS) (Malinová et al., 2011). INTRODUCTION 31 Stimulation of the residual enzyme Biotin replacement in biotinidase and multiple carboxylase deficiencies; hydroxycobalamine in defects of cobalamin metabolism and thiamine in thiamine-responsive MSUD deficiency are such examples of cofactor therapy (Barends et al., 2014). Some disorders result from a defect in enzyme protein folding and lead to conformational change. Pharmacological chaperones are small molecules which can recover the misfolded proteins by stimulating them to be folded. This fact has been used for the treatment of a group of patients with PKU by using BH4. BH4 acts as a chaperone for the same enzyme (Underhaug et al., 2012) Replacement of the missing enzyme Enzyme replacement therapy (ERT) is an approach to treating specific IEM, including LSDs, that replaces a deficient or absent enzyme (Parenti et al., 2015). ERT is based on the ability of most cells to uptake the exogenous enzyme through the mannose or mannose-6phosphate receptors present on their cell surface and pass it to lysosomes (Rome et al., 1979). ERT is available for Gaucher disease, Fabry disease, Pompe disease, and MPS types I, II, IV, and VI and is under development in other disorders (Desnick and Schuchman, 2012). The main disadvantage of the existing enzyme preparations that they do not cross the blood-brain barrier and, as a result, do not affect the central nervous system findings (Kishnani and Beckemeyer, 2014; Desnick and Schuchman, 2012). Transplantation of the cells, organs that have the potential to replace the missing enzyme, has been used as an authorized treatment for some IEM. Bone marrow transplantation (BMT) has successfully been used for MPS type I. BMT has the advantage of providing the missing enzyme to CNS as well as the other tissues. Liver transplantation is a choice of treatment of some cases of MMA and PA, UCDs, and some glycogen storage disorders (Boelens et al., 2009; Mazariegos et al., 2014). INTRODUCTION 32 Removal of the toxic substrate Branched-chain amino acids (leucine, isoleucine, and valine) accumulation in aminoacidopathies, as the levels of these toxic compounds, do not decrease rapidly in response to diet, it is necessary to force their removal to avoid/minimize associated neurological damage. To achieve this, the toxin can be removed directly (dialysis, for example), or medication can be administered to divert the metabolic pathway or provoke rapid excretion in the urine. However, hemodialysis or hemofiltration continues to be the fastest and effective method for toxic metabolites removing such as ammonia. Some medications are also being used orally or with intravenous infusions, to help with the excretion of the toxic substrate. (Brusilow et al., 1979), introduced the idea of treating UCD by using alternative biochemical pathways to eliminate excess nitrogen. This led to the use of oral sodium phenyl butyrate as a maintenance medication for disposal of excess nitrogen, intravenous sodium phenyl acetate (the active, metabolic product of sodium phenyl butyrate), and sodium benzoate, used in conjunction with arginine hydrochloride for emergency management of hyperammonemia (Enns et al., 2007; Brusilow, 1991). In situations where ammonia levels cannot be controlled by the above interventions, hemodialysis is used (Alfadhel et al., 2013). L–carnitine has proven its efficacy for the treatment of certain organic acidemias and diseases that affect mitochondrial metabolism. L–carnitine administration main functions are: first, to promote formation of organic acylcarnitines to re-establish the coenzyme, second is to bind to free organic acids so that they can be filtered and excreted effectively by the kidneys (Clarke, 2005). INTRODUCTION 33 Dietary therapy in IEM disorders. Diet therapy is the mainstay of treatment for several type of IEM, especially small molecule metabolic diseases, such as AA, organic acids, UCDs, carbohydrate metabolism defects, such as galactosemia and HFI, and energy metabolism defects, such as GSD and FAO defects as MCAD. The principal strategies of dietary therapy in IEM include restricting the offending substrates or metabolites and providing deficient products or alternative energy sources to by-pass the defective pathway. The main goal is to maintain healthy growth and development (Gambello and Li, 2018). Dietary recommendations for patients with IEM are based on or extrapolated from estimated requirements for healthy populations, including recommendations from the WHO/FAO. However, as IEM diets often differ in natural protein and energy intake from these recommendations, their impact on long term growth and body composition needs on-going assessment. Effective treatment requires an understanding of both the biochemistry of such defects and the individual nutritional requirements, to provide an adequate intake and maintain metabolic balance (Collins and Leonard, 1985). The approach to dietary therapy is specific to each metabolic disorder, but the principles are identical. Examples of IEM required nutritional intervention are listed in Table 3. INTRODUCTION 40 ► Classic Homocystinuria. Homocystinuria patients in Spain are not responsive to vitamin pyridoxine (B6). Methionine restricted diet with betaine, folic acid, vitamin B6, and B12 supplementations are used to control the biochemical abnormalities, especially to manage the plasma homocysteine concentrations and prevent complications, in particular, thromboembolism (Kaur et al., 1995). ► Maple syrup urine disease. Dietary leucine restriction, supplementation with isoleucine and valine, and frequent clinical and biochemical monitoring may help to manage MSUD patients (Chuang et al., 2006). 2. Disorders of carbohydrate metabolism Carbohydrate disorders show a wide range of clinical symptoms because of metabolism abnormalities. Carbohydrates are the main component of our diet, which are made up of long chains of simple sugar molecules. Normally cellular enzymes metabolize carbohydrates into glucose or simpler molecules. If an enzyme needs to metabolize specific sugar is missing, the sugar can accumulate in the body, causing health problems. Carbohydrate metabolic disorders result due to the defect in one or more enzymes involved in carbohydrate metabolism. ► Classic galactosemia. Classic galactosemia is caused by enzyme galactose-1-phosphate uridyltransferase (GALT) deficiency, manifests in the neonatal period, triggered by galactose intake, generally from breast milk, leading to cholestasis jaundice, liver failure, renal tubular dysfunction, sepsis, and cataracts. INTRODUCTION 41 The clinical symptoms of galactosemia can be controlled with nutritional therapy, mainly by galactose and lactose free diet. Infants should be fed a formula (e.g., soy formula) that contains trace levels of galactose or lactose. Continued restriction of dairy products in older children is recommended for galactosemia. However, studies have described that dietary restriction does not affect the long-term complications as psychomotor development delays, and motor abnormalities, possibly because of endogenous galactose production, which is independent of restricted dietary intake (Bosch, 2006). ►Hereditary fructose intolerance. HFI is caused by deficiency of the enzyme aldolase B which splits fructose-1-phosphate into dihydroxyacetone phosphate and glyceraldehyde and converts the triphosphates into glucose and lactate. Accumulation of fructose-1-phosphate inhibits both hepatic glycogenolysis and gluconeogenesis, hence inducing hypoglycemia, and results in depletion of adenosine triphosphate. This results in a number of disturbances, e.g. inhibition of protein synthesis and ultra-structural lesions, causing hepatic and renal dysfunction (Rake et al., 2006). Fructose is a natural monosaccharide found in many fruits, vegetables, and honey. The deficiency of enzyme fructose-1,6bisphosphate aldolase allows the accumulation of fructose-1-phosphate in the liver, kidney and small intestine resulting in metabolic inhibition of glycogen and glucose, thereby causing severe hypoglycemic condition (low sugar in the blood) in the human body. Nutritional treatment along with a fructose-free diet is effective to manage symptoms of HFI (Yasawy et al., 2009). INTRODUCTION 42 ► Glycogen storage diseases (GSD) A genetic defect in catabolic pathway of glycogen lead to develop GSD. Deficiency of enzymes involved in the glycogen metabolism result in progressive accumulation of glycogen in the liver and muscles. The most affected organ is the live (Adeva-Andany et al., 2016). Glycogen storage disease type I (GSD-I) also known as Von Gierke disease is an inherited defect due to glucose-6-phosphatase enzyme deficiency, it causes metabolic abnormalities in glycogen metabolism. Nutritional therapy may help to maintain blood glucose levels, to control hypoglycemia, and to provide optimal nutrition for growth and development. Dietary therapy improved survival of patients with GSD-I. Prognosis and occurrence of complications depend on the long-term metabolic control regarding life-threatening hypoglycaemia or lactic acidosis. GSD-I patients may be neurologically normal under adequate dietary treatment. However, because of recurrent severe hypoglycaemia they may also be mentally handicapped. Normal growth and pubertal development can be expected today (Mayatepek et al., 2010). The nutritional interventions include frequent daytime feedings, night-time intragastric continuous glucose infusion and oral uncooked corn starch may be necessary for the management of this disease. Liver function test must be monitored for the efficacy of dietary treatments (Froissart et al., 2011). In GSD III, a high protein diet is used to treated infants and children. Corn starch may already be introduced in the first year of life. Although fructose and galactose can be metabolized, the restrictions of so called simple/fast carbohydrates are a matter of debate. These simple sugars include glucose, galactose, lactose (galactose + glucose), fructose, sucrose (fructose + glucose), and maltodextrin. Fasting should be avoided, and for the overnight, a combination of snacks, frequent feeds, corn starch may be needed. Adolescents and adults have lower basic carbohydrate requirements. The recommended daily amount of protein is 25 % of the total caloric intake. A bedtime snack or an INTRODUCTION 43 overnight high protein formula may be prescribed for patients with myopathy (Kishnani et al., 2010). 3. Disorders of fatty acids oxidation metabolism. Nutrition management of all FAODs includes avoidance of fasting, aggressive treatment during illness, and supplementation of carnitine, if necessary. ► Dietary treatment of asymptomatic/symptomatic Very long chain acyl– CoA dehydrogenase deficiency (VLCADD), MCADD, and LCHADD. Asymptomatic newborns without elevated creatinine kinase (CK) and transaminase, continuation of breast milk is suggested. In MCADD, treatment comprises regular meals and avoidance of prolonged fasting periods (Spiekerkoetter et al., 2009b). In case of MCADD children and adults require regular meals and snacks during the day and before bed to prevent hypoglycemia and fatigue (Frazier, 2008)2. In LCHADD should be added a product with medium chain triglycerides (MCT) The primary goal of nutrition management of LC-FAOD is to limit long chain fat as a substrate for energy production both by preventing β-oxidation and catabolism and by limiting the amount of dietary long chain fat while still providing adequate nutrients for normal growth and development. Avoiding essential fatty acid (linoleic acid C18:2n6 and α-linolenic acid C18:3n3) deficiency is important, and the majority of long chain fat consumption should come from oils rich in essential fatty acids such instead of saturated long chain fatty acids as butter, fatty meats, etc. (Rohr and Calcar, 2008)3. In symptomatic patients with elevated CK or transaminase, the diet should contain greater amounts 2 http://gmdi.org/Resources/Nutrition-Guidelines/MCAD 3 http://gmdi.org/Resources/Nutrition-Guidelines/VLCAD INTRODUCTION 44 of MCT formula and less or no breast milk (Spiekerkoetter et al., 2003; II et al., 2018). Supplementation with specific oils such as walnut or flaxseed oil may be necessary to meet essential fatty acid requirements. Patients are at higher risk for becoming deficient in fat soluble vitamins and may require supplementation with a fat restricted diet, patients with LCFAODs are potentially at risk for inadvertently lowering their protein intake unless they specifically consume low fat, high protein foods (e.g., nonfat dairy, lean meats). An investigation of a higher protein diet in LCHADD patients showed a higher protein diet with less carbohydrates does not improve metabolic control, but may be beneficial for body composition and liver lipid content (Gillingham, 2015; II et al., 2018). Nutrition management of MADD is complex and requires a low fat (20–25% energy) and low protein diet to decrease excess intake of isoleucine, leucine, lysine, tryptophan, and valine (Angle and Burton, 2008). Patients even need to avoid MCT oil because β-oxidation of all fatty acid chain lengths is compromised (El-Gharbawy and Vockley, 2018). Additionally, patients require avoidance of fasting, adequate energy to prevent catabolism as well as supplementation with riboflavin and carnitine (Olsen et al., 2007; II et al., 2018) L-carnitine supplementation in FAOD was started because of low carnitine plasma concentrations found in many patients, suggesting secondary carnitine deficiency (Winter, 2003; II et al., 2018). 1.2 Body Composition Measurements The study of body composition allows us to know the proportions of the different principal components of the human body; in this way, its variation with age, growth, sports practice, and the various physiological and pathological conditions can be evaluated. Body composition can include estimates of percent body fat (BF) or fat mass (FM), fat free mass (FFM), lean body mass (LBM), bone mineral content (BMC), and total body water (TBW) (B. Heymsfield et al., 1997). There are different methods to measure body composition. INTRODUCTION 45 Anthropometrics could include measures of height, weight, and circumferences. In contrast, BF, FFM, and muscle mass are often predicted using methods such as a sum of skinfolds, Bioelectric Impedance Analysis (BIA) and dual-energy X-ray absorptiometry (DEXA). The human body can be assessed at five levels, depending on clinical concerns. Figure 6 shows the multi-compartmental or five level model of body composition. Figure 6: Multi-compartmental or Five levels body composition assessment Indirect measurement Indirect methods, including anthropometry and BIA, provide estimates or indices of body composition. Anthropometric. Anthropometric assessment is the set of measurements of the body (weight, height, circumferences, and skinfolds) at different ages and determines the different levels and degrees of nutritional status of an individual or a group. The measures are relatively simple, fast, and ECS: Extracellular solids, ECF: Extracellular fluids ECF: Extracellular fluids INTRODUCTION 46 economical, the anthropometric data are capable of reflecting changes in the nutritional intake produced in the long term and the results obtained should be evaluated compared with standard references according to the age and sex of the individual (Frisard et al., 2005). ► Weight establishes an indicator of body mass and volume, which is the most used and useful measure in pediatric practice. A variety of scales is available for weight measuring and should be calibrated regularly for accurate assessments of weight. ► Height measured easily with a variety of wall mounted equipment (Figure 7). Additional methods have been developed for predicting height when it cannot be measured directly, e.g., for the disabled or mobility impaired. Stature reflects skeletal growth; this has efficacy in the comparison of population groups or the long-term monitoring individual (Chumlea et al., 1994, 1998). Figure 7: Height stadiometer. INTRODUCTION 47 ► Body mass index (BMI) is a descriptive index of the body that expressed as weight divided by height squared (kg/m2). A significant advantage of BMI is the availability of an extensive national reference data and its established relationships with levels of body fatness, morbidity in children and adults, and besides mortality in latest adults (Organization; 2000; Chumlea; Guo,2000). High BMI percentage levels based on Centers for Disease Control and Prevention (CDC) BMI growth charts and changes in parameters of BMI curves in children are linked to significant levels of risk for comorbidity in pediatrics age and adult obesity (Guo et al., 2002, 2000). BMI is widely used to estimate body fat because it is inexpensive and straightforward. (Organization, 2000; Cole et al., 2000). ► Measurements of the circumferences (wrist, arm, waist, hip, thigh, and leg) give us complete information about body composition and BF distribution specially waist and hip measurement (Figure 8). Abdominal circumference, a centralized fat pattern is associated with the deposition of intra-abdominal and adipose tissue (Smith et al., 2001). Figure 8: Circumferences common body sites INTRODUCTION 48 ► Skin-fold measurements describe subcutaneous fat thickness in different body regions (Figure 9). Most national reference data available are for skinfolds at the triceps and subscapular locations. Skinfolds are particularly useful in monitoring changes in fatness in children because of their small body size, and most of the fat is subcutaneous. With the measurement of the four biceps, triceps, scapular, and suprailiac skinfolds, the greater or lesser degree of adiposity can be predicted (Brambilla et al., 1994; Bartrina et al., 2006). Figure 9: Skin folds measurements A: biceps and triceps, B: subscapular, C: suprailiac skin folds. Bio-electric Impedance Analysis (BIA). BIA is a commonly used method for estimating body composition FFM and FM. BIA measures the impedance or resistance to a small electrical current as it travels through the body’s water pool. BIA’s advantages include its portability and ease of use, relatively low cost, minimal participation required, and safety (not recommended for participants with a pacemaker), thus making it attractive for large-scale studies (Rush et al., 2006; Chumlea and Guo, 1994). INTRODUCTION 49 Direct measurements. Are methods for analysis from the atomic view through the cellular levels, such as neutron activation, isotope dilution, and total body counting. Total Body Water (TBW). TBW is easy to measure as it does not require undressing or any real physical participation. Water is the most abundant component in the body, and TBW volume is measured by isotope dilution. Water maintains a relatively stable relationship to FFM; therefore, measured water/isotope dilution volumes allow prediction of FFM and fat in weight individuals (Chumlea et al., 2002; Armstrong et al., 1997). Total Body Counting and Neutron Activation. Total body counting (also called whole-body counting) measures the amount of naturally radioactive potassium 40. As potassium is found almost entirely within cell bodies, measuring potassium can provide an estimate of body cell mass. FFM can then be estimated once total body potassium is known (Ellis, 2000). Neutron Activation involves high levels of neutron radiation exposure and has not been used in large-scale population research. Imaging method. Three major techniques are used for imaging of the body: computer tomography (CT), magnetic resonance imaging (MRI), and DEXA. Imaging methods measure a property of the body, such as its density, or describe amounts and distributions of skeletal, muscle, and adipose tissues via x-ray or magnetic imaging techniques. JUSTIFICATION 56 OBJECTIVES 57 3. OBJECTIVES The fundamental objectives of this work have been to study the body composition and the nutritional status of IEM patients followed in the Unit of Diagnosis and Treatment of Congenital Metabolic Diseases of the University Hospital Complex of Santiago de Compostela. 3.1 Main outcomes 1) Study of growth and development of the IEM Children by anthropometric and body composition evaluation. 2) Evaluate nutritional status in the IEM children by dietary intake record and nutritional biochemical biomarkers. 3.2 Secondary 1) Determine the risk of osteopenia and/or osteoporosis by bone mineral density measurement with DEXA in different body zone. 2) Analyse the effects of prolonged dietary restrictions on the state of micronutrients. 58 MATERIAL AND METHODS 59 4. MATERIAL AND METHODS 4.1 Patients Study Design This is a cross-sectional observational study that uses cases (patients with IEM) after the start of a dietary and/or medical treatment) carried out in the Unit of Diagnosis and Treatment of Congenital Metabolic Diseases during the period 2017-2019. A similar matched group by age and sex of 98 healthy children was collected during the same period. Population A total of 99 metabolic patients with age range 5 to 19 years old were studied. The collected data of the patients included: type of diagnosis, diagnosis time, sex, age (years), birth weight(kg) and height (cm), socioeconomic status and educational level of parents, anthropometric measurements (weight, height, BMI, circumferences (mid-upper arm, wrist, waist, hip, thigh, and calf circumferences), skinfolds (biceps, triceps, subscapular, and suprailiac), puberty stage, FM, FFM, bone densitometry of total body, lumbar spine and proximal femur by DEXA were performed. Each child underwent biochemical, nutritional evaluation. Dietary and pharmacological treatment and treatment compliance data were also included. Physical activity questionnaires were evaluated. The same measurements applied in sex and age-matched healthy controls. The project was explained to participating children and adolescents who met the inclusion criteria and none of the exclusion and to their parents or legal guardians. They were also provided with an information sheet related to the study. Once their participation was accepted, an informed consent approved by the Research Ethics Committee (Galicia CEIC Registration Code: 2017/310) was signed by the parents or legal MATERIAL AND METHODS 60 guardians and by children over 12 years of age. Appendix B: informed consent. Inclusion and Exclusion criteria In order to participate in the study, they had to meet the following criteria. Inclusion criteria • Children 5-19 years of age diagnosed with inherited metabolic disorders. • Informed consent to participate in the study will be signed by the parents or guardians of the children. The data will be collected in an encrypted manner in a database. Appendix B: informed consent. Exclusion criteria • Do not approve informed consent. • Have been diagnosed in the last year. • Have another associated disease that affects growth. • Be participating in a research project or less than three months that have participated. MATERIAL AND METHODS 61 Ethical aspects This study followed the Organic Law 3/2018, of December 5, on the Protection of Personal Data and guarantee of digital rights, and follow the fundamental principles established in the Declaration of Helsinki, in the Council of Europe Convention on Human Rights and Biomedicine, in the UNESCO Universal Declaration on Human Rights, and the requirements established in the Spanish legislation in the field of medical research, and will follow the rules of Law 14/2007 on Biomedical Research and Organic Law 15/1999, RD 1720/7007 for the protection of personal data, Biomedical Research, and adjusts to what is established in Law 31/1995, of November 8, on the Prevention of Occupational Risks, and in the Royal Decrees that develop it regarding risks related to exposure to biological agents. Appendix A. 4.2 Method Anthropometric assessment The following parameters are collected in the study. These measurements were performed by the same person (the researcher), which were repeated three times each to minimize both inter-observer and intra-observer bias. ►Weight was measured to the nearest 0.1kg while the child was wearing light clothing and no shoes, with the use of SECA 701 electronic medical scales with a class III digital display. They placed on the scale in a standard anatomical position without using any support point that could vary the measure. The measured values are converted to z-score according to the international reference values of the Centers for Disease Control and Prevention (CDC) (Kuczmarski, 2000). MATERIAL AND METHODS 62 ►Height was measured by using the Harpenden stadiometer, measuring 600-2100 mm, approved by the University of London Institute of Child Health. Height is measured while the child is standing without shoes, heavy outer garments, and hair ornaments. The back of the head, shoulder blades, buttocks and heels are touching the stadiometer. The head of the stadiometer is lowered so that the hair is pressed. Reading of the height should be done from eye level. The measured values were converted to z-score according to the international reference values of the World Health Organization (WHO)4. ►BMI: the Quetelet index (weight / size2) was calculated as kilogram per square metre (kg/m2), along with standardised scores and centiles, which was scored using the the international reference values of the World Health Organization (WHO)5. ►Body circumferences: measured with a flexible, not extensible Seca 201 tape, allows measuring circumferences with millimeter precision. For measuring, the tape is held at a right angle to the limb or body segment being measured, and the tape tension must be constant. This constant tension is achieved by ensuring that there are no gaps between the skin and the tape and maintains its place at the specified reference. Hold the tape box with the right hand and the end of the tape with the left. Passes the end of the tape around and takes the tip of the tape with the right hand, then holds both the end and the box. Apply enough tension to the tape with your right hand to keep it in position, while your left-hand goes under the box to retake the end. Now the tape outlines the segment to be measured; the middle fingers of both hands are free to precisely position the tape at the mark and orient it so that the zero is easily read. When recording the reading, the tester's eyes 4 https://www.who.int/growthref/who2007height for age 5http://www.euro.who.int/en/health-topics/disease-prevention/nutrition/a-healthy-lifestyle/bodymass-index-bmi MATERIAL AND METHODS 63 should be at the same level as the tape to avoid any error of parallelism between tape and limb or segment. All measures were converted to zscore according to the Spanish reference value for children's and adolescents /enkid study among the Spanish population (SerraMajem et al., 2002). The following sites have been measured: ▪ Mid-Upper Arm Circumference (MUAC): measured on the left arm left upper arm at the mid-point between the tip of the shoulder and the tip of the elbow (olecranon process and the acromion). At this level and with the arm relaxed, surrounded the tape without compressing the tissues. MUAC values were converted to z-score according to the Spanish reference value for children and adolescents /enkid study among the Spanish population (Serra-Majem et al., 2002). ▪ Wrist circumference: place the tape around the wrist above the ball joint of the ulna to determine wrist's circumference in cm. Body frame size can be determined measuring your wrist and comparing it to your height. ▪ Waist circumference: measurement is taken around the abdomen at the level of the umbilicus (belly button). Waist circumferences were converted to z-score according to the Spanish reference value for children’s and adolescents /enkid study among the Spanish population (SerraMajem et al., 2002). ▪ Hip circumference: the circumference was taken in the horizontal plane of the maximum level of the gluteal muscles, the individual remained standing and with the gluteal muscles relaxed. Hip circumferences were converted to z-score according to the Spanish reference value for children’s and adolescents /enkid study among the Spanish population (Serra-Majem et al., 2002). MATERIAL AND METHODS 64 ▪ Thigh circumference: the circumference of the thigh was taken with the individual in a biped position, distributing the weight on both feet, placing the tape 1cm under the buttock fold perpendicular to the longitudinal axis of the thigh. ▪ Calf circumference: was measured using inelastic tape with participants in the upright position at the maximum circumference at the plane perpendicular to the calf's longitudinal line. ►Skinfolds measurements: they were measured using Harpenden Analog Skin-Fold Caliper, Model Harpenden Skinfold Caliper, for Medical Research, which reports a compression of 10 gms/sq.mm, with a measurement range of up to 80 mm, in divisions of 0.2, being able to interpolate accurately up to 0.1 mm. The exact point where the skinfold is taken must be carefully located using anatomical marks before evaluation. In this study, measurements were made on the left side of the body. For the correct taking of the fold, it must be "pinched" in the previously marked place. Once the double portion of skin plus the underlying subcutaneous adipose tissue is "pinched," it is held under pressure between the thumb and index finger. Hold the caliper with your right hand, pressing to separate the branches and then applying them at a right angle to the direction of the fold and approximately one centimeter from the fingers. Later the pressure exerted on the equipment is released, wait two seconds, and proceed to take the reading in millimeters. Finally, the instrument is removed, and the fold is released. Four skinfolds have been measured and all converted to z-score according to Serra Spanish reference of enkid study (Serra-Majem et al., 2002): ▪ Triceps skinfold: pinch the subcutaneous fat at the triceps level on the back of the arm, 1 cm above the mark made for the brachial perimeter. Check that the skin-fold separates from the muscle. The caliper is applied horizontally over the mark with the right hand, holding the pinch with the left hand, for about 3 MATERIAL AND METHODS 65 seconds before recording it. Triceps values converted to z-score according to Serra Spanish reference of enkid study (SerraMajem et al., 2002). ▪ Biceps skinfold: it is taken at the same level as the triceps fold but on the front of the arm. A fold of skin and subcutaneous fatty tissue should be made along the longitudinal axis of the arm. Biceps values converted to z-score according to Serra Spanish reference of enkid study (Serra-Majem et al., 2002). ▪ Subscapular skinfold: locate the left scapular angle and make a mark. Pinch the subcutaneous fat 1 cm above and medial to the mark made, forming an angle of 45o concerning the vertebral column (the fold points to the contralateral elbow). The same procedure described for the triceps fold measurement will be followed. Subscapular values converted to z-score according to Serra Spanish reference of enkid study (SerraMajem et al., 2002). ▪ Suprailiac skinfold: pinch the subcutaneous fat at the level of the intersection of the mid-axillary line with the iliac crest. Oblique fold forward and down. Suprailiac values converted to z-score according to Serra Spanish reference of enkid study (Serra-Majem et al., 2002). Puberty stage The pubertal stage only in the IEM patients was assessed by a study physician. TheTanner scale describes the physical changes observed in the genitals, breast and pubic hair in both sexes. This internationally accepted scale classifies and divides pubertal changes into five stages ranging from child (I) to adult (V). For our study, we divided children and adolescents into two groups, prepubertal (Tanner I) and pubertal (Tanner II, III, IV and V). Body composition assessment Body composition (FM, muscle mass, and BMC) in addition to BMD was determined through dual-beam X-ray absorptiometry (lunar 72 RESULTS 73 5. RESULTS 5.1 General characteristics of sample Total sample: IEM patients and controls, general characteristics 197 subjects (including 99 IEM patients and 98 healthy matching controls) participated in this stuy. The females represented 57.6% and 51.1% in IEM patients and controls group respectively with no significant differences between the two groups. No significant difference in age stage between IEM patients and the healthy matched controls with age range between 5 and 19 years ( median age= 11.66 years) Tables 4 and 5. Table 4: Sample distribution by sex P significant < 0.05 Table 5: Sample distribution by age stages P significant < 0.05 Patients Controls Sex N % N % P Females 57 57.6 50 51.1 0.082 Males 42 42.4 48 48.9 0.074 Total 99 100 98 100 Patients Controls Age N % N % P 5 to 9 38 38.4 33 33.7 0.062 10 to 14 25 25.3 32 32.7 0.067 15 to 19 36 36.3 31 31.6 0.063 Total 99 100 98 100 RESULTS 74 In both groups of IEM patients and healthy controls, the presence of pregnancy-related diseases, hypertension, and diabetes in mothers during pregnancy, occurs only in 2% and 1% respectively, without significant differences. Chronic diseaes such as celiac disease, IEM, etc., are presented in 8% of IEM patient mothers and 4% in control mothers. Among these diseases, 5 of IEM patient mothers were diagnosed with IEM 3HPA and 2 hypermethioninemia MAT I/III mothers. Tables 6, 7 and 8. Table 6: Mothers diseases during pregnancy for IEM patients and controls Patients Controls Presence of disease N % N % P Pregnancy related diseases Yes 2 2 1 1 0.919 No 96 98 97 99 0.941 Total 98 100 98 100 Cronic disease Yes 8 8 4 4 0.024 No 90 92 94 96 0.039 Total 98 100 98 100 P significant < 0.05 Table 7: Frequency of disease in mothers during pregnancy for IEM patients and controls Patients Controls Disease N % N % p Diabetes 1 1 1 1 0.945 Hypertension 1 1 0 0 0.921 Non 96 98 97 99 Total 98 100 98 100 P significant < 0.05 RESULTS 75 Table 8: Frequency of cronic diseases in mothers of IEM patients and controls Patients Controls Disease N % N % p Celiac disease 1 1 1 1 0.931 Hypercholesterolemia 0 0 2 2 0.840 Hepatitis 0 0 1 1 0.838 Thyroid disease 2 2 0 0 0.645 IEM disease 5 5 0 0 0.042 Non 90 92 94 96 0.457 Total 98 100 98 100 P significant < 0.05 Total sample, IEM patients, general characteristics Most of the IEM patients of this study, 94% have been diagnosed early by NBS, patients diagnosed later represent only 6% of the total sample. Table 9. Table 9: Frequency of diagnosis time in IEM patients Thirteen IEM patients, 8 females and 5 males, had affected relatives, some of them with the presence of more than member in the same family. In total 21% (n=21) members in families of the 13 IEM patients are diagnosed with IEM.The highest percent of the disease observed in the brothers of the patients followed by mothers and sisters with 9% and 5% respectively. Tables 10, 11, 12, 13 and 14. Diagnosis time in patients N % Early diagnosis (newborn screening) 92 94 Late diagnosis 6 6 Total 98 100 RESULTS 76 Table 10: Frequency of family history of IEM in patietns N of patients % Patients with no family history of IEM 85 86.7 Patients with Family history of IEM 13 13.3 Total 98 100 Table 11: Frequency of IEM among patient’s family members Presence of IEM in patient’s family N % No IEM in family members 78 79 Family members with IEM 21 21 Total 99 100 Table 12: Who in the family have IEM Table 13: Frequency of IEM among patients’ families for sex Family member N % No one 78 79 Father 1 1 Mother 5 5 Brother 9 9 Sister 5 5 Others (cousins.) 1 1 Total 99 100 Patients Females Males Presence of IEM in patient’s family N % N % P No 49 86 37 88.1 0.000 Yes 8 14 5 11.9 0.003 Total 57 100 42 100 RESULTS 77 Table 14: Frequency of incidence of IEM in members of IEM patient family for sex Patients Females Males Family member N % N % P No one 44 77 34 81 0.000 Father 1 2 0 0 0.062 Mother 2 4 3 7 0.003 Brother 7 12 2 5 0.000 Sister 3 5 2 5 0.005 Others (cousins.) 0 0 1 2 0.063 Total 57 100 42 100 N: number of subjects, P significant at <0.05 According to the socio-economic level, in the IEM patient's, only 16% reach a high level, while 21% have a low level, and the rest represent 63% with a moderate level. Table 15. Table 15: Frequency of the socioeconomic status in IEM patients’ parents Socioeconomic Status level N % Low level 20 21 Moderate level 62 63 High level 16 16 Total 98 100 Regarding the level of education, 40% of the patient's parents have a medium level, while 51% represented those with low education levels, 9% of the patient's parents have a higher level of education. Table 16. Table 16: Frequency according to the level of education in IEM patients’ parents Education level N % Low level 50 51 Moderate level 39 40 High level 9 9 Total 98 100 RESULTS 78 Binary logistic regression analysis, with the dependent variable as a group (patients versus controls) and the independent variables sex, birth weight, study level of parents, and family history of the presence of IEM disorders, was conducted and the identified model (Likelihood Ratio: statistics= 90.787, p =0.001). In parents, at the study level, the positive coefficient indicated that an increase of one degree in the study level of parents increases the presence of healthy children. More family members with IEM recorded in patients’ group than controls. The logistic regression analysis identified two risk factors for this pathology: family history of IEM and parent study level. According to the results presented in the next table, family history IEM is expected to be almost five times more frequent in the IEM group compared with the controls. According to the parent study level, a high level of education is more prevalent in the control group than patients, and its 1.6 times higher in healthy controls group. Table 17. Table 17: Results of binary logistic regression analysis of IEM patients and controls Coefficient S.E. Wald P* OR Sex -0.196 0.314 0.390 0.533 0.822 Birth weight (kg) 0.283 0.316 0.800 0.371 1.327 Parents study level 0.467 0.255 3.354 0.017 1.595 Family history of IEM -3.000 1.040 8.317 0.004 5.089 R2 =0.597 SE: standard error, OR: odds ratio, *significant at p<0.05. Dependent variable :(0: patients, 1: controls). Independent variables: sex (1: female;2: male), Birth weight (kg), Parent study level (low, moderate, high), family history of IEM (1: yes,2: no). RESULTS 79 General characteristics of intermediary metabolism disorders AA metabolism disorders were the most prevalent in IEM patients, representing 77.8% (n=77) followed by carbohydrate metabolism disorders (CHD) with 12.1% (n=12) of the total IEM patient's sample, while fat metabolism disorders 10.1% (n=10). Table 18 and Figure 13. Table 18: Frequency of intermediary metabolism disorders in IEM patients Patients N % Amino acids disorders 77 77.8 Fat disorders 10 10.1 Carbohydrate disorders 12 12.1 Total 99 100 Figure 13: Disease category for all IEM patients RESULTS 80 For the AAs meabolism disorders, the most incidence was HPA (12 females and nine males), and PKU (14 females to 11 males), five males and four females have hypermethioninemia (14.7% vs. 9.3%, respectively), four MSUD comparing to two males and two MMA females. In fat metabolism disorders, patients with SCADD were all females. In CHD, three males and two females were presented with HFI disease, two females, and one male with classic galactosemia. Table 19. Table 19: Specific disorders diagnoses in IEM patients enrolled in the study for sex HPA: Hyperphenylalaninemia, MCADD: Medium chain acyl-CoA dehydrogenase deficiency, MSUD: Maple syrup urine disease, OTC: Ornithine transcarbamylase, PKU: Phenylketonuria, SCADD: Short chain acyl-CoA dehydrogenase deficiency. Patients Females Males N % N % Amino acids disorders HPA 12 27.9 9 26.5 PKU 14 32.5 11 32.4 Hypermethioninemia MAT I/III 4 9.3 5 14.7 MSUD 4 9.3 2 5.8 Tyrosinaemia type 1 2 4.7 2 5.8 Glutaric aciduria type 1 3 7 3 8.8 Citrulinemia type I 1 2.3 0 0 OTC deficiency 0 0 1 2.9 3-Hydroxy-3-methylglutaric aciduria 0 0 1 2.9 Methylmalonic aciduria 2 4.7 0 0 Nonketotic hyperglycinaemia 1 2.3 0 0 Carbohydrate disorders and defects transport of carbohydrates Classic galactosaemia 2 28.6 1 16.7 Hereditary fructose intolerance 2 28.6 3 50 Glycogen storage disease 2 28.6 2 33.3 Glucose transporter 1 deficiency 1 14.2 0 0 Fat oxidation disorders MCADD 3 42.9 3 100 SCADD 4 57.1 0 0 RESULTS 81 5.2 Anthropometric characteristics Total sample: IEM Patients and Controls Anthropometric characteristics. 56.6% of the IEM pateints and 65.3% of the controls were having normal weight (BMI 5-85th percentile n=56 vs. n=64), 35.4% of patients and 30.6% of the controls were overweight /obesity (BMI >85th percentage n=35 vs. n=30) among this 25 IEM patients showed BMI > 95th percentage while only six controls in the same percentile. 8.1% of IEM patients and 4.1% of the controls were underweight (BMI < 5th percentile, n=8 vs. n=4)8. Table 20. Table 20: Distribution of BMI percentage according to WHO International standards in patients and controls. BMI: body mass index, N: number of subjects, p significant at <0.05. Table 5.18 shows that IEM patients are more frequent with high BMI z-score, nine IEM patients, having z-score between 1.036 to1.64, in the z-score 1.645 to3.09 we founded 24 IEM patients in this category, while 2 of the IEM patients recorded a z-score ≥ 3.090, eight IEM patients have underweight with z-score < -1.645. In the controls group, only 4 of them recorded a low z-score < -1.645, among the highest BMI 8 http://www.euro.who.int/en/health-topics/disease-prevention/nutrition/a-healthy-lifestyle/body-massindex-bmi Patients Controls p BMI N % N % Underweight (P<5th) 8 8.1 4 4.1 0.039 Normal weight (p 5-85th) 56 56.6 64 65.3 0.000 Overweight and obesity (p>85th) 35 35.4 30 30.6 0.000 Total 99 100 98 100 RESULTS 88 Intermediary metabolism disorders anthropometric characteristics. Overweight was more prevalence in patients with AA metabolism disorders with 36.4% (n=28) and underweight with 6.5% (n=5) respectively followed by CHD disorders with 33.3% (n=4) overweight patients and 16.7% (n=2) with underweight, while in FAO disorders presented 30% (n=3) patients with overweight and one with underweight.10 Tables 29. Table 29: Prevalence of the degree of adiposity according to WHO International standards in intermediary metabolism disorders BMI category AA disorders Fat disorders CH disorders N % N % N % Underweight (P<5th) 5 6.5 1 10 2 16.7 Normal weight (P 5-58th) 44 57.1 6 60 6 50 Overweight and obesity (p>85th) 28 36.4 3 30 4 33.3 Total 77 100 10 100 12 100 N: number of subjects, AA: Amino acids, CH: Carbohydrate Twenty-eight patients with AA disorders are reported with z-score > 1.036 in which two of them were with the highest z-score value > 3.090, CHD come next with four patients recording z-score > 1.036, but no one has a z-score > 3.090, only two patients with FAO presented with > 1.036. Table 30. 10 http://www.euro.who.int/en/health-topics/disease-prevention/nutrition/a-healthy-lifestyle/bodymass-index-bmi RESULTS 89 Table 30: Distribution of BMI z-score in intermediary metabolism groups. Category z-score AA disorders Fat disorders CH disorders N % N % N % - 3.090 to ≤ -1.645 5 6.5 1 10 2 16.7 -1.645 to ≤ 1.030 44 57.1 6 60 6 50 1.036 to ≤ 1.28 8 10.4 1 10 0 0 1.645 to 3.09 18 23.4 2 20 4 33.3 >3.090 2 2.6 0 0 0 0 Total 77 100 10 100 12 100 N: number of subjects, AA: Amino acids, CH Carbohydrate. In assessment of body composition for intermediary metabolism disorders, weight mean z-score was no significant differ for each group. Patients with CH disorders have the lowest height z-score mean (-1.173 ±1.037, p = 0.002) followed by AA metabolism disorders with z-score mean value (-0.268 ±1.176, p = 0.000). BMI presented a higher z-score with significant differences in AA and FAO metabolism disorders from controls (p <0.05). No significant differences observed in body circumferences except in waist circumferences, which presented significant differences in AA metabolism disorders (p <0.05). In skinfolds measurements, biceps wase higher and significant in AA metabolism disorders (p =0.000). Table 31. RESULTS 90 Table 31: Means of body composition z-score measurements among intermediary metabolism disorders. Patients Controls Anthropometric Disorders N Mean ± SD N Mean ±SD P Weight z-score AA 77 0.469 ±1.12 98 0.486 ±0.86 0.913 FAO 10 0.935 ±0.98 20 0.297 ±0.94 0.136 CHD 12 -0.349±1.39 20 0.248 ±0.76 0.179 Height z-score AA 77 -0.267±1.18 98 0.148 ±0.95 0.012 FAO 10 0.711±1.16 20 -0.13 ±0.82 0.061 CHD 12 -1.173±1.04 20 -0.03 ±0.92 0.007 BMI z-score AA 77 0.662 ±1.22 98 -0.373±0.95 0.000 FAO 10 0.64 ±1.33 20 -0.68 ±0.98 0.014 CHD 12 -0.033±1.73 20 -0.379±0.83 0.447 Mid-arm circumference AA 77 -0.242±1.14 98 -0.373±0.99 0.417 z-score FAO 10 -0.15±1.3 20 -0.68±0.98 0.287 CHD 12 -0.9±1.45 20 -0.379±0.83 0.339 Waist circumference AA 77 -0.075±1.35 98 -0.588±1.11 0.008 z-score FAO 10 -0.285±1.57 20 -0.794±1.09 0.382 CHD 12 0.052±1.2 20 -0.363±0.69 0.251 HIP circumference AA 77 -0.635±1.08 98 -0.821±0.88 0.319 z-score FAO 10 0.219±1.19 20 -1.014±0.83 0.080 CHD 12 -1.16±1.23 20 -0.778±0.84 0.443 Biceps z-score AA 77 0.778±1.57 98 0.288±1.44 0.036 FAO 10 0.241±2.44 20 0.109±1.28 0.672 CHD 12 0.443±1.52 20 0.787±125 0.726 Triceps z-score AA 77 0.131±1.25 92 0.015±1.4 0.547 FAO 10 -0.215±1.59 12 0.022±1.33 0.708 CHD 12 -0.052±1.28 15 0.298±1.03 0.514 Subscapular zscore AA 77 1.1±2.38 98 0.578±1.79 0.106 FAO 10 1.293±1.95 20 0.82±1.84 0.811 CHD 12 0.817±2.15 20 0.792±1.46 0.930 Suprailiac z-score AA 77 1.38±1.86 98 1.03±1.66 0.201 FAO 10 1.293±1.95 20 0.82±1.84 0.565 CHD 12 1.363±2.04 20 1.176±1.12 0.755 AA: amino acids, BMI: body mass index, CHD: carbohydrate disorders, FAO: fatty oxidation disorders, N: number of participants, SD: standard deviation, p significant at <0.05. RESULTS 91 Binary logistic regression indicates that height, biceps, triceps, and waist are significant predictors of status of IEM patients and controls (Chi-Square= 34.414, df =4 and p =0.000). All the four predictors “explain” 22% of the variability of patients and controls. Height, biceps, triceps, and waist are significant at the 5% level [waist Wald= 8.449, p =0.004 (p <0.05); height (Wald= 9.878, p =0.002); biceps (Wald= 8.240, p =0.004); and triceps (Wald= 11.843, p =0.000). Height showed a positive coefficient indicated that controls were higher than patients. The negative coefficients (-0.941) in biceps indicated that patients have higher biceps skinfold than controls, an indirect indication of fat mass, which is higher in patients. Triceps were higher in controls than IEM patients (coefficient = 1.336), while waist with negative coefficient = -0.605. The odds ratio (OR) for the waist is 0.546, height 1.672, triceps 3.804, and biceps 0.390. The model predicted correctly at 67.5%. Among all these variables both triceps and height consider the most risk factors in predicting patients and controls, in which patients are 1.7 times lower in height than controls and 3.8 times lower in triceps circumference than controls. Table 32. Table 32: Summary of Logistic Binary Regression Analysis for anthropometric variables in patients and with IEM and controls Coefficient S.E. Wald p OR Height z-score 0.514 0.164 9.878 0.002 1.672 Biceps z-score -0.941 0.328 8.240 0.004 0.390 Triceps z-score 1.336 0.388 11.843 0.000 3.804 Waist z-score -0.605 0.208 8.449 0.004 0.546 R2=0.220 S.E.: standard error, OR: odd ratio, p significant at <0.05. Dependent variable :(0: patients, 1: controls). Independent variables: height, biceps, triceps, waist. RESULTS 92 5.3 Body Composition Assemment. Total sample: IEM patients and controls body composition assessment. The study of the DEXA, there were no significant differences in muscle mass and fat mass measurements and in bone density in the lumbar spine L2-L4 between IEM patients and controls, in the other hand significant differences in the total body density between IEM patients and controls with low mean value in IEM patients (0.89±0.95, 1.6±1.5, p =0.001) as shown in Figure 15. The femur in the three sites (neck, trochanter, and ward) was significantly lower in IEM patients (p <0.05). Table 33. Table 33: Means of body composition z-score measured by DEXA in patients and controls Patients Controls Muscle mass z-score mean ± SD mean ± SD p 0.09±1.6 0.29±1.3 0.317 Fat mass z-score 0.26±1.8 -0.07±1.3 0.159 BMD total z-score 0.89±0.95 1.6±1.5 0.001 Lumbar spine L2-L4 z-score 0.65±0.69 0.8±0.64 0.117 Femur neck z-score 0.45±0.76 0.67±0.75 0.044 Femur trochanter z-score 0.5±0.98 0.84±0.89 0.012 Femur ward z-score -0.21±0.71 0.04±0.8 0.023 BMD: bone mineral density, SD: standard deviation, p significant at <0.05. RESULTS 93 Figure 15: Mean total body bone mineral density in IEM patients and Controls Our results demonstrated that BMD total body z-score mean was significantly different between IEM patients females and controls females (0.91 ±0.96 vs. 1.4±0.96, p= 0.008, respectively). Reduced BMD total body z-score means were also founded in patients males when compared to controls males (0.88±0.96 vs. 1.9±2, p = 0.003, respectively). Femure BMD z-score mean was lower in IEM patients males than in controls males in three measured sites with significant differences between the two groups (p< 0.05). Femur neck was 0.19±0.61 in patients males versus 0.52±0.62 in controls males, femur trochanter with 0.19±0.64 in patients males and 0.59±0.63 in controls males, femur ward was -0.39±0.73 in patients males versus 0.033±0.62 in controls males. No significant differences regarding muscle mass and fat mass were founded between males and females of the two studied groups. Table 34. RESULTS 94 Table 34: Means of body composition z-score measured by DEXA according to sex in patients and controls Patients Controls Sex N Mean ±SD N Mean ±SD p Muscle mass z-score F 57 0.45±1.3 50 0.42±0.91 0.896 M 42 -0.41±1.8 48 0.16±1.6 0.118 Fat mass z-score F 57 -0.18±1.5 50 -0.22±0.89 0.871 M 42 0.86±2.1 48 0.09±1.7 0.063 BMD total z-score F 57 0.91±0.96 50 1.4±0.96 0.008 M 42 0.88±0.96 48 1.9±2 0.003 BMD Lumbar spine L2-L4 F 57 0.75±0.61 50 0.83±0.64 0.522 z-score M 42 0.51±0.77 48 0.77±0.65 0.096 BMD Femur neck F 57 0.64±0.81 50 0.81±0.84 0.275 z-score M 42 0.19±0.61 48 0.52±0.62 0.017 BMD Femur trochanter F 57 0.73±1.1 50 1.08±1.02 0.099 z-score M 42 0.19±0.64 48 0.59±0.63 0.004 BMD Femur ward F 57 -1.4±0.69 50 0.08±0.95 0.161 z-score M 42 -0.39±0.73 48 0.033±0.62 0.045 BMD: Bone Mineral Density, F: Female, M: Male, N: Number, SD: standard deviation, p significant at <0.05, Table 35 shows the correlation between anthropometric and BMD of all skeletal sites in patients and controls. In patients, Pearson’s correlation coefficient (r) was significant (p <0.01) and positive among bone variables (BMD of the spine, femoral trochanter, femoral ward, and femur), with weight. BMI founded to be significant (p <0.01) and positive with BMD total body and BMD of the spine, and significant (p <0.05) and positive correlation with (femoral trochanter, femoral ward, and femur). Total body BMC, BMD spine were positively correlated with age (p <0.01), while a positive significant (p <0.05) founded with femur and trochanter. On average, the weight seemed to have a strong correlation with BMD in IEM patients. RESULTS 95 In controls group Pearson’s correlation coefficient (r) was significant (p <0.01) and positive among bone variables (BMD total, BMD of the spine, femur trochanter, femur ward, and femur neck), with weight, and BMI. Age founded to be significant (p <0.01) and positive with BMC and with BMD total and femur (p <0.05). Table 35: Pearson’s correlation coefficient between bone mineral density (BMD and anthropometrics variables in IEM patients and controls Patients BMD BMC BMD BMD BMD BMD total L2-L4 Femur trochanter Ward Age in years r Pearson -0.153 0.834** -0.261** -0.252* -0.233* -0.142 Weight z-score r Pearson 0.865** 0.184 0.784** 0.723** 0.477** 0.409** BMI z-score r Pearson 0.618** 0.110 0.528** 0.517* 0.477* 0.247* Controls Age in years r Pearson 0.209* 0.547** -0.192 -0.208* -0.183 -0.107 Weight z-score r Pearson 0.920** 0.235* 0.519** 0.769** 0.329** 0.435** BMI z-score r Pearson 0.807** 0.164 0.483** 0.439** 0.379** 0.422** **. Correlation is significant at 0.01. *. Correlation is significant at 0.05. BMD: bone mineral density, BMC: bone mineral content, BMI: body mass index. RESULTS 96 Binary logistic regression analysis, with the dependent variable as a group (patients versus controls) and the independent variables BMD total body, spine, and femur were conducted and the identified model (Chi-square=20.609, p = 0.000). The logistic regression analysis identified BMD total as risk factors for this pathology. According to the results presented in Table 5.26, the BMD total is expected to be almost 1.9 times higher in controls than the patients IEM group. The other two variables show no significant relation. Table 36. Table 36: Results of binary logistic regression analysis of patients with IEM and controls R2=0.220 S.E.: standard error, OR: odd ratio, P significant at <0.05, BMD: bone mineral density Dependent variable :(0: patients, 1: controls). Independent variables: BMD total; BMD spine L2-L4; BMD femur. According to the determination of BMD by DEXA, osteopenia risk in both groups was detected with significant differences between the patients and the controls group (p = 0.036), no history of fracture was recorded in any of the children in patients and controls. In the patient's group, 33 (33.3%) presented osteopenia risk and seven with sever reduced BMD <-2.5 labled as osteoporosis risk (7.1%). Among the controls group, 20 (20.4%) presented osteopenia risk, and no one has a very low bone density (z < -2.5). Table 37. Table 37: Prevalence of osteopenia and osteoporosis risk for IEM patients and controls. BMD L2-L4 z-score groups Normal Osteopenia risk Osteoporosis risk p % (n) % (n) % (n) Patients 58.6 (58) 33.3 (33) 7.1 (7) 0.036 Controls 79.6 (78) 20.4 (20) 0 BMD: Bone Mineral Density, p significant at <0.05. Coefficient S.E. Wald p OR BMD total z-score 0.636 0.167 14.504 0.000 1.888 BMD spine L2-L4 -0.386 0.301 1.646 0.200 0.680 BMD femur 0.217 0.203 1.134 0.287 1.242 RESULTS 97 Intermediary metabolism disorders body composition assessment. According to DEXA, measured variables BMD for the intermediary metabolic disorders showed that the FM z-score was higher among AA disorders than in controls (p <0.05). Total body BMD z-score was lower in AA metabolism disorders patients than in controls (0.833±0.92 vs. 1.66±1.35, p= 0.000). AA metabolism disorders, according to the femur, three sites bone density (femur neck, trochanter, and ward) founded to be significantly lower from reference controls (p <0.05). Table 38. Table 38: Means of body composition z-score measured by DEXA in intermediary metabolism disorders Patients Controls Diseases category N Mean ±SD N Mean ±SD p Muscle mass AA 77 0.023±1.39 98 0.297±1.26 0.383 z-score FAO 10 1.515±2.3 20 0.24±1.16 0.110 CHD 12 -0.687±1.39 20 0.046±1.24 0.254 Fat mass AA 77 0.315±1.83 98 -0.071±1.35 0.026 z-score FAO 10 -0.021±2.34 20 -0.35±1.22 0.676 CHD 12 0.162±1.65 20 0.086±0.73 0.940 BMD total AA 77 0.833±0.92 98 1.66±1.56 0.000 z-score FAO 10 1.38±0.76 20 1.493±1.06 0.780 CHD 12 0.863±1.26 20 1.639±1.63 0.213 BMD Spine L2-L4 AA 77 0.641±0.68 98 0.88±0.64 0.321 z-score FAO 10 1.024±0.59 20 0.935±0.78 0.818 CHD 12 0.38±.71 20 0.88±0.76 0.206 BMD femur Trochanter AA 77 0.491±095 98 0.84±0.89 0.013 z-score FAO 10 1.06±1.16 20 0.45±1.25 0.753 CHD 12 0.11±0.85 20 0.49±1.15 0.350 BMD Femur neck AA 77 0.424±0.79 98 0.67±0.75 0.039 z-score FAO 10 0.528±1.33 20 0.867±0.76 0.473 CHD 12 0.26±0.56 20 0.36±0.89 0.733 BMD femur Ward AA 77 -0.252±0.71 98 0.04±0.8 0.013 z-score FAO 10 0.283±0.66 20 -0.25±1.2 0.229 CHD 12 -0.33±0.59 20 -0.33±0.84 0.994 AA: amino acids, BMD: Bone mineral density, CHD: carbohydrate disorders, FAO: fatty acid oxidation, p significant at <0.05. RESULTS 104 5.5 Dietary Contribution and Correlation with Body Composition. Total sample: IEM patients and controls dietary contribution and correlation with body composition. Protein intake significantly very low among children with IEM comparing to the control (55.75±21.23, 75.67±4.61, p = 0.000), also for protein-energy, protein %, and protein total per kg of body weight ( p <0.05). See Figures 16 and 17. Both groups are no different in dietary intake of fat and energy. Patients consume more carbohydrates with total intake in grams per day (234.57 ±119.76, vs. 201.79±39.26, p =0.013) with a percent of carbohydrate energy from the total calories intake 88.3% compared to healthy with 52.46%. Table 48. Among the nutrient intake, there was no significant differences in cholesterol intake between groups with low mean recorded in IEM patients than controls (172.97 vs. 179.86). Minerals intake present equal consumption between IEM patients and controls, potassium, calcium, magnesium, phosphorus, iron, selenium, and zinc. A lower significant difference in sodium intake with a mean (1111.54 mg vs. 1397.71 mg, p =0.000) in patients and controls respectively, flour intake was higher in patients (mean=10.96 mg) comparing to controls (mean = 5.21 mg, p =0.042). Vitamins intake of folate is differing significantly with high intake in IEM patients versus controls (283.3±155.3 μg vs. 226.13±165.3, p = 0.015), fat-soluble vitamins D, K, and E have no significant differences. In contrast, vitamin A was higher in IEM patient's, with a mean intake of (527.28 vs. 440.7, p = 0.047). Table 48. RESULTS 105 Figure 16: Mean dietary total protein intake in IEM patients and controls. Figure 17: Mean dietary protein natural intake in IEM patients and controls. RESULTS 106 Table 48: Means of dietary intake/day in IEM patients and controls Patients Controls Dietary intake/day Mean ±SD Mean ±SD P Protein total (g) 55.75±21.23 75.67±14.61 0.000 Energy Protein (Kcal) 216.19±83.63 302.59±58.57 0.000 Energy Protein % 13.95±4.65 20.06±3.77 0.000 Fat Total (g) 47.96±20.21 47.73±10.86 0.802 Energy Fat (Kcal) 418.37±158.69 430.40±97.42 0.628 Energy Fat % 26.91±7.43 28.08±5.22 0.290 CH Total (g) 234.57±119.76 201.79±39.26 0.013 Energy CHO (Kcal) 893.84±265.69 808.76±157.07 0.008 Energy CHO % 88.34±5.68 52.46±5.81 0.093 Energy total (Kcal) 1566.93±399.72 1540.83±224.77 0.487 Energy (Kcal/kg) 40.99±17.06 43.09±19.49 0.489 Protein total (g/kg) 1.41±0.65 2.08±0.89 0.000 Cholesterol (mg) 172.97±104.99 179.86±102.62 0.647 Sodium (mg) 1111.54±346.58 1397.71±593.01 0.000 Potassium (mg) 1814.71±503.78 1782.63±598.12 0.688 Calcium (mg) 717.46±231.69 673.46±325.80 0.284 Magnesium (mg) 190.26±74.40 182.71±86.32 0.517 Phosphorus (mg) 879.07±273.92 874.15±323.69 0.117 Iron (mg) 11.84±7.22 10.17±7.35 0.910 Fluor (μg) 10.96±23.01 5.21±14.56 0.042 Selenium (μg) 64.57±29.67 66.91±34.97 0.617 Zinc (mg) 7.18±3.42 6.88±3.57 0.557 Folate (μg) 283.30±155.27 226.13±165.30 0.015 Vitamin B12 (μg) 5.91±6.78 6.61±15.41 0.685 Vitamin A (μg) 527.28±296.93 440.7±301.52 0.047 Vitamin D (μg) 1.49±0.82 1.56±1.23 0.624 Vitamin K (mg) 71.66±63.73 75.77±94.87 0.724 Vitamin E (mg) 4.51±3.52 4.16±3.47 0.491 CH: Carbohydrate, p significant at <0.05 Ficant at <0.05. RESULTS 107 Protein intake shows a non-significant positive correlation with weight and BMI. However, protein intake has a negative correlation with FM. Still, it was non-significant; for muscle mass, a significant positive relation between protein intake and muscle mass (r =0.234) which explain that the increase of protein intake will increase the muscle mass), p <0.05. Intake of natural protein was significant correlated with muscle mass (r= 0.217, p = 0.003). Energy intakes have a significant positive correlation with body weight (r = 0.206, p <0.05). Table 49. Table 49: Correlation of protein dietary intake with weight, BMI, FM, and muscle mass for IEM patients. Weight BMI Fat mass Muscle mass Protein total (g) 0.079 0.139 -0.073 0.234* Protein natural (g) 0.045 0.069 -0.048 0.217** Energy total (kcal) 0.206* 0.130 0.099 0.088 **. Correlation is significant at the 0.01 level *. Correlation is significant at the 0.05 level. BMI: body mass index Table 50 shows the correlation between dietary intake and anthropometric variables in control group. Protein intake shows a nonsignificant positive relationship with weight and BMI, muscle mass, and a negative correlation with fat mass. For muscle mass, a significant positive association between protein intake and muscle mass (r= 0.187) (the increase of protein intake will increase the muscle mass). Energy intake has a significant positive correlation with BMI (r= 0.235, p <0.05). Table 50: Correlation of protein dietary intake with weight, BMI, Fat mass, and muscle mass for Controls. Weight BMI Fat mass Muscle mass Protein total (g) 0.121 0.098 -0.129 0.187 Protein natural (g) 0.121 0.098 -0.129 0.187 Energy total(kcal) 0.176 0.235* 0.138 0.131 *. Correlation is significant at the 0.05 level. BMI: body mass index RESULTS 108 Both total protein and natural protein intake were positively and significantly correlated with total body BMD (r = 0.186, r= 0.254, p <0.01) respectively. A significant positive Pearson coefficient revealed between protein intake with trochanter (r= -0.177, p = 0.014) and ward (r= 0.171, p =0.018). Also, protein natural was positives and significant relation with femur (r = 0.213, p =0.003), trochanter (r= 0.257, p =0.000) and ward (r= 0.233, p =0.001). Both calcium and vitamin D intake have no relation with BMD. Table 51. Table 51: Pearson correlation of dietary intake and BMD in the total sample **. Correlation is significant at the 0.01 level. *. Correlation is significant at the 0.05 level. BMD: bone mineral density. When separating the sample, the IEM patients BMC was correlated positively with total protein, total energy, and negatively with the phosphorous intake (p < 0.01). At the same time, BMD has only a positive significant correlation with natural protein intake. BMD of the ward and trochanter had a positive relation with natural protein intake (p < 0.05). Both calcium and vitamin D intake have no relation with bone density in IEM patients. Table 52. BMD BMD L2-L4 BMD femur BMD trochanter BMD Ward total Protein total 0.186** 0.047 0.130 0.177* 0.171* Protein natural 0.254** 0.140 0.213** 0.257** 0.233** Calcium 0.036 0.015 0.051 0.012 0.087 Vitamin D 0.050 0.071 0.015 0.005 0.033 RESULTS 109 Table 52: Pearson correlation of dietary intake and BMD in IEM patients BMD BMC BMD BMD BMD BMD total Tot g L2-L4 trochanter Femur Ward Protein total (g) r Pearson 0.179 0.454** 0.013 0.147 0.065 0.198* Protein natural(g) r Pearson 0.264** 0.196 0.156 0.264** 0.208* 0.287** Calcium (mg) r Pearson 0.036 0.107 0.015 0.012 0.051 0.087 Phosphorus (mg) r Pearson -0.019 -0.363** -0.124 -0.097 -0.159 -0.007 Vitamin D (μg) r Pearson 0.050 0.031 0.071 0.005 0.015 0.033 **. Correlation is significant at the 0.01 level. *. Correlation is significant at the 0.05 level. BMD: bone mineral density, BMC: bone mineral content Among the controls group BMC, BMD trochanter, BMD femur, and BMD ward not correlated significantly with total protein, total energy, and phosphorous intake, protein natural, calcium, and vitamin D intake. BMD has only a positive significant correlation with energy intake. BMD of the spine was having a significant positive relation with vitamin D intake (r =0.263, p =0.011). Table 53. Table 53: Pearson correlation of dietary intake and BMD in controls BMD total BMC total g BMD L2-L4 BMD trochanter BMD femure BMD ward Protein total (g) r Pearson 0.046 0.144 0.045 0.080 0.013 0.032 Protein natural (g) r Pearson 0.046 0.144 0.045 0.080 0.013 0.032 Calcium (mg) r Pearson 0.076 0.142 0.094 0.080 0.106 0.163 Phosphorus (mg) r Pearson -0.068 -0.104 -0.079 -0.017 -0.029 -0.003 Vitamin D (μg) r Pearson 0.036 0.021 0.263* 0.082 0.147 0.122 *. Correlation is significant at the 0.05 level. BMD: bone mineral density, BMC: bone mineral content RESULTS 110 Binary logistic regression indicates that protein total, protein natural, total energy intake, and protein /kg body weight are significant predictors of the status of IEM patients and controls [Chi-Square= 113.119, df= 6 and p =0.000 ( p <0.05)]. All four predictors explain 59.6% of the variability of patients and controls. Dietary intake of protein and energy are significant at the 5% level [protein total intake, Wald=3.795, p =0.040; protein natural intake, Wald= 3.797, p =0.040]; [energy total, Wald= 5.364, p =0.021]; and [protein total kg, Wald= 5.830, p =0.000]. The odds ratio (OR) for protein total is 0.860, and protein natural is 1.614, energy total 0.997, and protein total/kg 2.666. The model correctly predicted 78.8% of IEM patients and 88% of controls, giving an overall percentage correct prediction rate of 83.2%. Both protein total/kg, and protein natural considers the most important factors in predicting patients and controls, in which IEM patients are 2.7 times lower in protein total g/kg than controls and 1.6 times lower in protein natural than controls, coefficients negative for carbohydrate and energy intake indicate that patients consumed more carbohydrate and energy than controls, but they consumed more fat as the coefficient is positive. Table 54. Table 54: Results of binary logistic regression analysis of patients with IEM and controls R2=0.220 S.E.: standard error, OR: odd ratio; p significant at <0.05, CH: carbohydrate Dependent variable :(0: patients, 1: controls). Independent variables: protein total, protein natural, fat total, CH total, energy total, protein total (g/kg). Coefficient S.E. Wald P OR Protein total (g) 0.088 0.286 3.795 0.040 0.860 Protein natural (g) 0.015 0.269 3.797 0.040 1.614 Fat total (g) 0.022 0.018 1.584 0.208 1.023 CH total (g) -0.006 0.004 2.250 0.134 0.994 Energy total (kcal) -0.003 0.001 5.364 0.021 0.997 Protein total (g/kg) 0.726 0.300 5.830 0.016 2.666 RESULTS 111 Intermediary metabolisn disorders dietary contribution and correlation with BMD. According to intermediary metabolism disorders, daily dietary intake varies with category. The lowest significant protein intake observed in AA metabolism disorders 50.38 g/d (p =0.000), also significant difference presented with the highest amount of fat consumed by CH metabolism disorders patients (60.48 g/d vs. 44.58 g/d in controls), while FAO metabolism disorders patients consumed less fat than controls (38.09, p =0.033). CH intake was significantly higher in AA metabolism and CHD disorders comparing to controls. Total energy intake per day was not significantly differ between intermediary groups and controls. Table 55. Table 55: Means of dietary intake/day in intermediary metabolism disorders. Patients Controls Dietary intake/day Diseases category N Mean ±SD N Mean ±SD p Protein total (g) AA 77 50.38±15.9 98 75.67±14.6 0.000 FAO 10 83.29±27.26 20 74.55±14.97 0.753 CHD 12 67.21±24.9 20 79.17±15.3 0.821 Protein natural (g) AA 77 32.18±23.57 98 75.67±14.6 0.000 FAO 10 83.29±27.26 20 74.55±14.97 0.658 CHD 12 67.21±24.9 20 79.17±15.3 0.835 Protein (g/kg) AA 77 1.23±0.45 98 2.08±0.89 0.000 FAO 10 2.03±0.58 20 2.297±0.95 0.432 CHD 12 2.08±1.02 20 1.99±0.64 0.421 Fat total (g) AA 77 47.67±18.11 98 47.73±10.86 0.954 FAO 10 38.09±13.11 20 49.04±9.27 0.033 CHD 12 60.48±32.24 20 44.58±9.3 0.002 CH total(g) AA 77 237.63±127.95 98 201.79±39.26 0.012 FAO 10 243.73±94.66 20 209.92±41.22 0.654 CHD 12 216.12±78.46 20 199.31±34.55 0.004 Energy total (Kcal) AA 77 1541.75±386.65 98 1540.83±224.77 0.945 FAO 10 1695.64±480.22 20 1587.58±237.04 0.854 CHD 12 1678.12±406.34 20 1516.69±200.7 0.027 Energy (Kcal/kg) AA 77 39.34±16.39 98 43.09v19.49 0.843 FAO 10 42.4712.89 20 49.12±17.88 0.895 CHD 12 52.48±22.56 20 38.67±13.37 0.756 AA: amino acid, CHD: carbohydrate disorders, FAO: fatty acid oxidation, P significant at <0.05. RESULTS 112 When studying the relationships between dietary intake and BMD in patients with IEIPM, daily dietary intake of protein natural founded to be correlated to bone density with positive and significant relation with p< 0.01 in BMD total and BMD trochanter, and with p < 0.05 with BMC, spine, femur, and ward. Both protein and energy total intake correlated with BMC with positive significant and seemed to be higher in protein (r=0.61 vs. r=0.339), respectively. A negative correlation between BMC and BMD trochanter and phosphorus intake, which means high dietary intake of this mineral, could cause low bone density as high dietary intake of phosphorus will deplete calcium from bones, especially if accompanied with low calcium intake. Table 56. Table 56: Pearson correlation of dietary intake and BMD in patients with IEIPM BMD BMC BMD BMD BMD BMD total tot g L2-L4 Femur Ward trochanter Protein total (g) r Pearson 0.168 0.610** 0.034 0.036 0.126 0.052 Protein natural (g) r Pearson 0.296** 0.227* 0.257* 0.256* 0.274* 0.295** Calcium (mg) r Pearson 0.117 0.143 0.083 0.162 0.257* 0.206 Phosphorus (mg) r Pearson -0.095 -0.336** -0.203 -0.288* -0.111 -0.276* Vitamin D (μg) r Pearson 0.008 0.083 0.027 0.001 0.066 0.011 **. Correlation is significant at the 0.01 level. *. Correlation is significant at the 0.05 level. IEIPM: Inorn error of intermediary protein metabolism, BMD: bone mineral density, BMC: bone mineral content. RESULTS 113 Adherence to dietary treatment in PKU patient The sample consists of 26 patients with PKU (12 females, 14 males) with age range 6-18 years old (12.15 4.35). Several clinical phenotypes are distinguished according to the levels of Phe at the diagnosis: Classical PKU: Phe > 20 mg / dl (> 1,200 μmol / L). In our sample, we have sixteen patients with classic PKU, ten with mild-moderate PKU. Eleven patients are treated with BH4. Table 57. Table 57: General characteristics of PKU patients BMI z-score mean was significantly different in the total sample between females and males, classic PKU patients presented a significant difference among sex with the lowest BMI was recorded in females. Table 58. Mean age 12.15 ± 4.35 Sex (Female/Male) 12 F / 14 M Phenotype Classic 16 (61.5%) Mild/Moderate 10 (28.5%) Treatment with BH4 11 (42.3%) Mean weight (kg) 43.22 ±16.23 Mean height (m) 154.94 ±10.16) BMI (Kg/m2) 21.22 ±4.75 Underweight 7 (26.9%) Normal weight 16 (61.5%) Overweight 3 (11.5 %) RESULTS 120 Table 62: Mean of biochemical blood analysis for IEM patients and controls Patients Controls Blood variable Mean± SD Mean± SD P Prealbumin (mg/dL) 22.98±5.59 22.66±4.56 0.667 RBP (mg/dL) 3.51±0.97 3.72±2.35 0.421 Protein total (g/dL) 7.27±0.35 7.21±0.37 0.283 Albumin (g/dL) 4.64±0.214 4.69±0.207 0.798 Calcium (mg/dL) 9.77±0.33 9.76±0.29 0.771 Zinc (μg/dL) 101.69±17.63 103.3±12.44 0.468 Selenium (μg/L) 76.37±18.01 85.46±8.01 0.000 Iron (μg/dL) 89.8±33.19 89.55±34.81 0.967 Ferritin (ng/mL) 38.01±27.64 40.48±38.93 0.620 Transferrin (mg/dL) 291.08±43.01 264.34±46.96 0.685 Vitamin A (mg/dL) 0.39±0.14 0.36±0.11 0.325 Vitamin D (ng/mL) 23.86±11.58 22.33±6.8 0.272 Vitamin E (mg/dL) 1.69±2.01 4.38±4.81 0.003 Vitamin k (ng/dL) 0.91±1.45 0.32±0.26 0.003 Folate (ng/mL) 17.82±11.41 8.99±3.79 0.000 VitaminB12 (pg/mL) 1621.4±8030.14 577.95±221.79 0.046 Cholesterol (mg/dL) 157.86±26.44 165.6±27.12 0.047 TG (mg/dL) 84.2±43.39 59.77±27.57 0.000 HDL (mg/dL) 56.92±51.51 57.71±11.55 0.888 LDL (mg/dL) 87.78±19.65 107.88±110.95 0.126 P significant at <0.05, HDL: High Density Lipoprotein, LDL: Low Density Lipoprotein, RBP: Retinol Binding Protein, TG: Triglyceride RESULTS 121 According to the reference values for serum vitamin D, the frequency of blood analysis values shows a high deficiency and insufficiency of the vitamin in both groups of IEM patients and controls (44.4%, 44.6), respectively. No defect of B12 observed in both groups. Two IEM patients versus seven control presents with vitamin A deficiency. Vitamin K was low in five patients and eleven controls. More normal values were present in controls too. Vitamin A with average values was more frequent in controls than IEM patients with significant differences (p = 0.021). Table 63. Table 63: Frequency of blood analysis for vitamins according to reference values p significant at < 0.05. Selenium deficiency presented in 36 IEM patients (36.4%) while in controls only one child has selenium deficiency (p = 0.000) and more controls are within normal values than IEM patients (n=88 vs. n=62). Iron and transferrin normal values are more frequent in controls than Patients Controls N % N % p Vitamin D (ng/mL) deficiency 5 5.1 2 2.2 0.456 insufficient 39 39.4 39 42.4 0.888 recommended 55 55.6 51 55.4 0.954 Folate (ng/mL) low 2 2 3 3.3 0.745 normal 53 53.4 85 92.4 0.000 high 44 44.4 4 4.3 0.000 Vitamin B12 (pg/mL) low 0 0 0 0 0.945 normal 96 97 92 100 0.756 high 3 3 0 0 0.256 Vitamin A (mg/dL) low 2 2 7 7.6 0.315 normal 31 1 53 57.6 0.021 high 1 1 0 0 0.756 Vitamin E (mg/dL) low 1 1 0 0 0.843 normal 13 13.1 14 15.2 0.542 high 20 20.2 45 48.9 0.030 Vitamin K (ng/dL) low 5 5.1 11 12 0.038 normal 21 21.2 51 55.4 0.002 high 5 5.1 0 0 0.621 RESULTS 122 patients with a significant difference; TG serum values are higher while cholesterol is lower in IEM patients than in controls (157.86 mg/dL vs. 165.6 mg/dL, p = 0.047). Table 64. Table 64: Frequency of blood analysis for nutrients and minerals according to reference value RBP: Retinol binding protein, TG: Triglyceride, p significant at <0.05. Patients Controls N % N % p Selenium (μg/L) low 36 36.4 1 4.3 0.000 normal 62 62.6 88 95.7 0.026 high 1 1 0 0 0.845 TG (mg/dL) low 0 0 1 1 0.832 normal 90 90 90 97.8 0.945 high 9 9 1 1.1 0.027 Cholesterol (mg/dL) low 10 10.1 1 1.1 0.026 normal 89 89.9 91 98.9 0.712 high 0 0 0 0 0.945 Zinc (μg/dL) low 1 1 0 0 0.888 normal 93 93.9 91 98.9 0.768 high 5 5.1 1 1.1 0.712 RBP (mg/dL) low 30 30.3 24 26.1 0.546 normal 68 68.7 61 66.3 0.658 high 1 1 1 1.1 0.921 Prealbumin (mg/dL) low 0 0 32 34.8 0.002 normal 32 32.3 60 65.2 0.003 high 67 67.7 0 0 0.000 Calcium (mg/dL) low 0 0 0 0 0.941 normal 99 100 92 100 0.765 high 0 0 0 0 0.955 Ferritin (ng/mL) low 1 1 3 3.3 0.845 normal 93 94 87 94.6 0.801 high 0 0 0 0 0.955 Iron (μg/dL) low 3 3 5 5.4 0.765 normal 47 47.5 85 92.4 0.004 high 1 1 2 2.2 0.895 Transferrin (mg/dL) low 1 1 2 2.2 0.865 normal 27 27.3 47 51.1 0.034 high 3 31.3 10 10.9 0.057 RESULTS 123 Binary logistic regression analysis, with the dependent variable groups (patients versus controls) and the independent variables serum triglyceride, serum folate, and serum selenium, was conducted identified with significant model (chi-square = 75.519, p =0.000). The logistic regression analysis identified three risk factors for this pathology: serum triglyceride, serum folate, and serum selenium level. According to the results, the overall variables predicted correctly at 76.7%. They founded that serum selenium level is expected to be almost 1.54 times higher in the control group compared with the IEM patients. In contrast, serum folate is expected to be 0.86 times lower in the control group than in patients, while serum triglyceride is expected to be lower in controls than the patients. Table 65. Table 65: Results of binary logistic regression analysis of patients with IEM and controls Coefficient S. E Wald P OR Serum Triglycerides -0.018 0.006 9.901 0.002 0.982 Serum Folate -0.146 0.032 20.815 0.000 0.864 Serum Selenium 0.042 0.015 7.702 0.006 1.043 R2=0.220, p significant at < 0.05. S.E.: standard error, OR: odd ratio, Dependent variable :(0: patients, 1: controls). Independent variables: serum triglyceride, serum folate, serum selenium. Biochemical and haematological markers in intemediary metabolism disorders. In AA metabolism disorders a significant difference was founded in serum transferrin which was lower in AA metabolism disorders patients than in controls (298.68±39.62 vs. 577.95±221.79 mg/dL, p = 0.039), cholesterol which was also lower in AA metabolism disorders (156.82±25.47 vs. 165.6±27.12 mg/dL, p = 0.043). At the same time, TG was higher in patients with AA metabolism disorders than controls (81.14±39.24 vs. 57.71±27.57 mg/dL, p = 0.000). Among FAO metabolism disorders ferritin was significantly lower in FAO metabolism patients compared to controls (65.33±45.41 vs. 92.00±20.72 ng/dL, p = 0.024), TG was higher in the FAO metabolism RESULTS 124 patients than in controls (74.2±44.2 vs. 58.08±23.42, p = 0.032 mg/dL), in the other hand HDL was lower in FAO metabolism patients than in controls (42.8±10.71 vs. 57.08±9.45mg/dL, p = 0.025). High serum TG level founded in CHD patients than in controls (111.92±86.96 vs. 70.00±29.01 mg/dL, p = 0.041), while LDL was lower in CHD metabolism patients than controls (86.00±20.07 vs. 155.08±185.36, mg/dL, p = 0.043). Table 66. Table 66: Mean of biochemical blood analysis for intermediary metabolism disorders. Patients Controls Disorders Mean± SD Mean± SD P Prealbumin (mg/dL) AA 23.18±5.1 22.66±4.6 0.485 FAO 23.1±7.7 23.92±4.52 0.764 CHD 21.58±6.86 24.15±4.89 0.399 RBP (mg/dL) AA 3.6±0.94 3.7±2.4 0.701 FAO 3.00±1.16 3.75±0.75 0.082 CHD 3.42±0.99 5.31±5.68 0.139 Protein total (g/dL) AA 7.26±0.44 7.24±0.46 0.914 FAO 7.3±0.48 7.42±.52 0.892 CHD 7.58±0.67 7.31±0.48 0.746 Albumin (g/dL) AA 4.84±0.37 4.9±0.29 0.257 FAO 4.8±0.42 5.00±0.01 0.914 CHD 4.83±0.39 4.92±0.28 0.811 Ferritin (ng/mL) AA 33.62±23.19 40.49±38.93 0.061 FAO 65.33±45.41 92.00±20.72 0.024 CHD 45.18±25.55 38.08±24.89 0.268 Transferrin (mg/dL) AA 298.68±39.62 577.95±221.79 0.039 FAO 247.00±52.33 288.75±26.58 0.091 CHD 264.13±45.84 290.08±24.89 0.107 Cholesterol (mg/dL) AA 156.82±25.47 165.6±27.12 0.043 FAO 171.4±14.18 170.25±23.43 0.894 CHD 153.25±37.18 169.00±30.86 0.061 TG (mg/dL) AA 81.14±39.24 57.71±27.57 0,000 FAO 74.2±44.2 58.08±23.42 0.032 CHD 111.92±86.96 70.00±29.01 0.041 HDL (mg/dL) AA 59.38±56.86 57.71±11.55 0.787 FAO 42.8±10.71 57.08±9.45 0.025 CHD 52.36±20.69 60.17±10.24 0.258 LDL (mg/dL) AA 86.16±19.08 107.88±110.9 0.142 FAO 110.2±13.06 101.58±22.84 0.446 CHD 86.00±20.07 155.08±185.36 0.043 AA: amino acids, CHD: carbohydrate disorders, FAO: fatty acids disorders, HDL: high density lipoprotein, LDL: low density lipoprotein, RBP: retinol binding protein, TG: Triglyceride. P significant at < 0.05. RESULTS 125 Vitamin K among AA metabolism disorders patients compared to controls with a lower concentration (0.91±1.45 vs. 0.32 ±0.26 mg/dL, p = 0.033). High vitamin B12 serum level founded in AA metabolism disorders patients (1895.69±909.25 vs. 577.95±221.79 pg/mL, p = 0.021). A significant high folate level in AA metabolism disorders patients compared to controls (20.33±11.56 vs. 9.00±3.81 ng/dL, p = 0.000). Selenium serum level was lower in AA metabolism disorders patients than in controls (74.55±18.68 vs. 85.46±8.01 μg/L, p = 0.011). FAO metabolism disorders observed with significant low iron (56.01 vs. 92.01 μg/dL), vitamin E (1.04 vs. 4.38 mg/dL) and vitamin D (15.9 vs. 20.92 ng/mL). No significant differences recorded in CHD. Table 67. RESULTS 126 Table 67: Means of serum blood vitamins and minerals analysis for intermediary metabolism disorders. Patients Controls Disorders Mean± SD Mean± SD P Vitamin A (mg/dL) AA 0.39±0.14 0.36±0.11 0.391 FAO 0.34±0.12 0.36±0.11 0.451 CHD 0.33±0.11 0.36±0.11 0.431 Vitamin D (ng/mL) AA 25.36±12.14 22.33±6.80 0.062 FAO 15.9±7.32 20.92±4.23 0.045 CHD 20.83±6.97 21.00±4.95 0.745 Vitamin E (mg/dL) AA 3.69±2.00 4.38±4.81 0.812 FAO 1.04±0.29 4.38±4.81 0.003 CHD 3.98±3.2 4.38±4.8 0.745 Vitamin k (ng/dL) AA 0.91±1.45 0.32±0.26 0.033 FAO 0.21±0.42 0.32±0.26 0.455 CHD 0.31±0.25 0.32±0.26 0.741 Folate (ng/mL) AA 20.33±11.56 9.00±3.81 0.000 FAO 10.4±5.85 10.75±4.71 0.878 CHD 8.33±3.94 9.46±3.48 0.712 VitaminB12 (pg/mL) AA 1895.69±909.25 577.95±221.79 0.021 FAO 648.7±168.5 658.67±374.8 0.939 CHD 672.00±214.74 728.46±329.34 0.514 Calcium (mg/dL) AA 9.78±0.33 9.76±0.29 0.853 FAO 9.75±0.27 9.84±0.27 0.745 CHD 9.75±0.38 9.92±0.26 0.565 Zinc (μg/dl) AA 101.6±16.9 103.3±12.44 0.455 FAO 101.05±0.12 104.67±11.51 0.544 CHD 99.75±23.85 108.00±13.74 0.268 Selenium (μg/L) AA 74.55±18.68 85.46±8.01 0.011 FAO 83.03±5.56 84.75±10.74 0.245 CHD 82.14±13.87 88.15±7.91 0.192 Iron (μg/dL) AA 94.69±33.12 89.55±34.81 0.423 FAO 56.01±2.83 92.01±0.27 0.035 CHD 70.14±26.14 85.31±24.41 0.212 AA: amino acids, FAO: fatty acid oxidation, CHD: carbohydrate, P significant at <0.05) DISCUSSION 127 6. DISCUSSION 6.1 Anthropometric Characteristics. This study shows significant findings in body composition measurements of IEM patients, such as reduced height, increased waist circumferences, increased prevalence of overweight/obesity, and increased FM. An important finding of this study is that IEM patients have significantly lower height z-scores than controls (z-score mean= - 0.28±1.23 vs. 0.16±0.93), with the lowest z-score recorded was -2.94. In this study, we found that height is lower in males patients comparing to controls males. This agrees with a study of Wilcox et al., in which they reported a reduced height in both sexes of patients with IEIPM compared to controls (Wilcox et al., 2005). CHD presented the lowest height value z-score (z-score mean= -1.173±1.04) with a significant difference from the control group, followed by the AA group. Panis et al., showed in children aged 3-17 years old with classic galactosemia on dietary treatment a significantly decreased mean height z-score (p< 0.001) (Panis et al, 2004). Reduced height and weight-for-height in childhood and early adolescence have been reported in treated classic galactosemia patients aged from 2 weeks to 37 years by Waggoner et al., (Waggoner et al., 1990). In the same line, Schweitzer et al. showed decreased height and weight in patients with classic galactosemia aged from 9 months to 33 years (Schweitzer et al., 1993). Pronicka et al., showed in children with HFI, aged 3-20years old, height deficiency with the lowest recorded z-score was -2.6. Those patients were followed a diet restricted in fructose, but 6 of them have a daily fructose intake above the recommended (2 g/day). A height deficit of HFI patients could be due to the presence of trace amounts of fructose in the diet or to quantitative and qualitative dietary deficiencies that are a side effect of diet elimination (Pronicka et al., 2007). Several authors have reported reduced height as common problems in GSD patients. Height was significantly reduced in patients with GSD I and III, with a median age of 11 years old compared to DISCUSSION 128 controls (Dos Santos, B. B., et al., 2017). Also, height was significantly reduced in the European Study (ESGSD I) for patients with GSD I (ESGSD I), median age 10.4 years (Rake et al., 2007). Regarding AA disorders, multiples early studies showed differences in body composition parameters to children with PKU, MMA, PA, and UCD (Batshaw et al., 2014; Hauser et al., 2011; Evans et al., 2017). Growth outcomes in MMA were poor in height, and body composition showed a significantly increased percentage of FM (Manoli et al., 2016). Evans et al. found that patients with MMA/PA had the lowest median height and weight z-scores (Evans et al., 2017). In contrast, patients with UCD presented normal weight but decreased linear growth. The differences in dietary patterns could explain this fact. UCD patients consumed a diet with restriction of total proteins and, consequently, higher CH and lipids Although MMA/PA is consuming a diet with restriction of a natural protein, but not total protein in the total caloric value (Batshaw et al., 2014). Reduced height z-scores were reported in PKU patients (AldámizEchevarría et al., 2014; Couce et al., 2015). Other studies found similar weight and BMI z-scores in PKU and controls (Allen et al., 1995; Adamczyk et al., 2011; Belanger-Quintana and Martínez-Pardo, 2011). However, it was also reported that children with PKU weight more than control (White et al., 1982; Scaglioni; Giovannini et al., 2007). One study on children with PKU showed lower weight than controls (Dobbelaere et al., 2003). These differences could be due to the type of PKU, complaints to treatment, and the number of dietary products with phenylalanine restriction consumption and small cohorts with limited statistical power. According to the FAO metabolism disorders, our study recorded the highest weight z-score comparing with control. Few reports have studied obesity in FAO, and most of them only documented the problem as a trend rather than a statistically significant issue without exploring its consequences. The diet regimen in FAO disorders based on reduced fat and diet that applied to prevent hypoglycemia by increased food consumption. This diet records a tendency to overweight; this has been DISCUSSION 129 observed in several studies with MCADD (Derks et al., 2006). But normal growth and weight have been recorded in other studies in MCADD patients (Wilcken et al., 2009). Although IEM patients trend for increased, we found no differences in weight and BMI z-scores mean between patients and controls, probably in relation to that more percentage of patients are within the highest and lowest percentage than controls. In order to know adiposity, we could evaluate BMI; in this study, the WHO BMI percentage and z-scores were used to assess overweight and obesity in children. 25.65% of IEM patients having a BMI percentage above 85th when compared to controls, only 6.3% having BMI above 95th. AA and FAO disorders presented a significant higher BMI z-score comparing to controls. In CHD, no significant difference in BMI zscore means and controls; however, 4 out of 12 patients with CHD tend to have higher BMI percentile >85th. The prevalence of overweight and obesity was higher in PKU patients aged 10–16 years than controls of similar age (Rocha, J. C., et al., 2012). Couce et al., showed that BMI z-score in PKU patients had a high value in 37 patients (26.24 %) [25 (67.6 %) were overweight, and 12 (32.4 %) were obese (z-score ≥2)] (Couce et al., 2016). On the other hand, the PKU population on a low phenylalanine diet trend of becoming overweight and obese is very similar to the healthy UK population (Robertson et al., 2013). According to FAO matbolism disorders, increased BMI z-score in our studied patient appears to be due to the high FFM z-score was presented. Multiple studies founded increased proportion of overweight in prepubertal patients (Derks et al., 2006; Iafolla et al., 1994). High rates of excess weight (overweight 28%; obesity 40%) were found among patients with GSD, 2 of 5 patients with GSD III and IXa/b were overweight, while 5 out of 6 with GSD Ib and 10 out of 14 with GSD Ia had excess weight (overweight or obesity), (Dos Santos, B. B., et al., 2017). Chen et al., found a high frequency of obesity in a study of 13 GSD I patients treated with uncooked cornstarch (Chen et al., 1993). Appendix 232 304 Operadores de equipos ópticos y electrónicos 305 Profesionales en navegación marítima 306 Profesionales en navegación aeronáutica 307 Técnicos en edificación, seguridad en el trabajo y control de calidad 31 TÉCNICOS DE LAS CIENCIAS NATURALES Y DE LA SANIDAD 311 Técnicos de las ciencias naturales y profesionales auxiliares asimilados 312 Técnicos de sanidad 313 Diversos técnicos de sanidad no clasificados en rúbricas anteriores 32 TÉCNICOS EN EDUCACIÓN INFANTIL, INSTRUCTORES DE VUELO, NAVEGACIÓN Y CONDUCCIÓN DE VEHÍCULOS 321 Técnicos en educación infantil y educación especial 322 Instructores de vuelo, navegación y conducción de vehículos 33 PROFESIONALES DE APOYO EN OPERACIONES FINANCIERAS Y COMERCIALES 331 Profesionales de apoyo en operaciones financieras y algunas operaciones comerciales 332 representantes de comercio y técnicos de venta 34 PROFESIONALES DE APOYO A LA GESTIÓN ADMINISTRATIVA 341 Profesionales de apoyo de la gestión administrativa, con tareas administrativas generales 342 Profesionales de carácter administrativo de aduanas, de tributos y asimilados que trabajan en tareas propias de las administraciones públicas 35 OTROS TÉCNICOS Y PROFESIONALES DE APOYO 351 Consignatarios y agentes en la contratación de mano de obra 352 Técnicos especialistas de las Fuerzas de Seguridad y detectives privados 353 Profesionales de apoyo de promoción social 354 Profesionales del mundo artístico, del espectáculo y de los deportes 355 Auxiliares laicos de las religiones 4 EMPLEADOS DE TIPO ADMINISTRATIVO 4G EMPLEADOS DE TIPO ADMINISTRATIVO 40 EMPLEADOS EN SERVICIOS CONTABLES, FINANCIEROS, Y DE SERVICIOS DE APOYO A LA PRODUCCIÓN Y AL TRANSPORTE 401 Auxiliares contables y financieros 402 Empleados de registro de materiales, de servicios de apoyo a la producción y al transporte 41 EMPLEADOS DE BIBLIOTECAS, SERVICIOS DE CORREOS Y ASIMILADOS 410 Empleados de bibliotecas, servicios de correos y asimilados 42 OPERADORES DE MÁQUINAS DE OFICINA 421 Taquígrafos y mecanógrafos 422 Grabadores de datos Appendix 233 43 AUXILIARES ADMINISTRATIVOS SIN TAREAS DE ATENCIÓN AL PUBLICO NO CLASIFICADOS ANTERIORMENTE 430 Auxiliares administrativos sin tareas de atención al público no clasificados anteriormente 44 AUXILIARES ADMINISTRATIVOS CON TAREAS DE ATENCIÓN AL PÚBLICO NO CLASIFICADOS ANTERIORMENTE 440 Auxiliares administrativos con tareas de atención al público no clasificados anteriormente 45 EMPLEADOS DE TRATO DIRECTO CON EL PÚBLICO EN AGENCIAS DE VIAJE, RECEPCIONISTAS Y TELEFONISTAS 451 Empleados de información y recepcionistas en oficinas 452 Empleados de agencias de viajes, recepcionistas en establecimientos distintos de oficinas y telefonistas 46 CAJEROS, TAQUILLEROS Y OTROS EMPLEADOS ASIMILADOS EN TRATO DIRECTO CON EL PÚBLICO 460 Cajeros, taquilleros y otros empleados asimilados en trato directo con el público 5 TRABAJADORES DE LOS SERVICIOS DE RESTAURACIÓN, PERSONALES, PROTECCIÓN Y VENDEDORES DE LOS COMERCIOS 5H TRABAJADORES DE LOS SERVICIOS DE RESTAURACIÓN Y DE SERVICIOS PERSONALES 50 TRABAJADORES DE LOS SERVICIOS DE RESTAURACIÓN 501 Cocineros y otros preparadores de comidas 502 Camareros, bármanes y asimilados 503 Jefes de cocineros, de camareros y asimilados 51 TRABAJADORES DE LOS SERVICIOS PERSONALES 511 Auxiliares de enfermería y asimilados 512 Trabajadores que se dedican al cuidado de personas y asimilados (excepto auxiliares de enfermería) 513 Peluqueros, especialistas en tratamiento de belleza y trabajadores asimilados 514 Trabajadores que atienden a viajeros y asimilados 515 Mayordomos, ecónomos y asimilados 519 Otros trabajadores de servicios personales 5J TRABAJADORES DE LOS SERVICIOS DE PROTECCIÓN Y SEGURIDAD 52 TRABAJDORES DE SERVICIOS DE PROTECCIÓN Y SEGURIDAD 521 Guardias civiles 522 Policías 523 Bomberos 524 Funcionario de prisiones 525 Guardias jurados y personal de seguridad privado Appendix 234 529 Otros trabajadores de los servicios de protección y seguridad 5K DEPENDIENTES DE COMERCIO Y ASIMILADOS 53 DEPENDIENTES DE COMERCIO Y ASIMILADOS 531 Modelos de moda, arte y publicidad 532 Encargado de sección dentro de un comercio y asimilados 533 Dependientes y exhibidores en tiendas, almacenes, quioscos y mercados 6 TRABAJADORES CUALIFICADOS EN LA AGRICULTURA Y EN LA PESCA 6L TRABAJADORES CUALIFICADOS EN LA AGRICULTURA Y EN LA PESCA 60 TRABAJADORES CUALIFICADOS EN ACTIVIDADES AGRÍCOLAS 601 Trabajadores cualificados por cuenta propia 602 Trabajadores cualificados por cuenta ajena en actividades agrícolas 61 TRABAJADORES CUALIFICADOS EN ACTIVIDADES GANADERAS 611 Trabajadores cualificados por cuenta propia en actividades ganaderas 612 Trabajadores cualificados por cuenta ajena en actividades ganaderas 62 TRABAJADORES CUALIFICADOS EN OTRAS ACTIVIDADES AGRARIAS 621 Trabajadores cualificados por cuenta propia en actividades agropecuarias 622 Trabajadores cualificados por cuenta propia en actividades forestales y asimilados 623 Trabajadores cualificados por cuenta ajena en actividades agropecuarias 624 Trabajadores cualificados por cuenta ajena en actividades forestales y asimilados 63 PESCADORES Y TRABAJADORES CUALKIFICADOS EN ACTIVIDADES PISCÍCOLAS 631 Pescadores y trabajadores cualificados por cuenta propia en actividades piscícolas 632 Pescadores y trabajadores cualificados por cuenta ajena en actividades piscícolas 7 ARTESANOS Y TRABAJADORES CUALIFICADOS DE LAS INDUSTRIAS MANUFACTURERAS, LA CONSTRUCCIÓN, Y LA MINERÍA, EXCEPTO LOS OPERADORES DE INSTALACIONES Y MAQUINARIA 7M TRABAJADORES CUALIFICADOS DE LA CONSTRUCCIÓN, EXCEPTO LOS OPERADORES DE MAQUINARIA 70 ENCARGADOS DE OBRA Y OTROS ENCARGADOS EN LA CONSTRUCCIÓN 701 Encargados y jefes de equipo en obras estructurales de la construcción 702 Jefes de taller y encargados de trabajadores de acabado de edificios 703 Encargados de pintores, empapeladores y asimilados 71 TRABAJADORES EN OBRAS ESTRUCTURALES DE CONSTRUCCIÓN Y ASIMILADOS 711 Albañiles y mamposteros Appendix 235 712 Trabajadores en hormigón armado, enfoscadores, ferrallistas y asimilados 713 Carpinteros (excepto carpinteros de estructuras metálicas) 714 Otros trabajadores de las obras estructurales de construcción 72 TRABAJADORES DE ACABADO DE CONSTRUCCIONES Y ASIMILADOS; PINTORES Y OTROS ASIMILADOS 721 Revocadores, escayolistas y estuquistas 722 Fontaneros e instaladores de tuberías 723 Electricistas de construcción y asimilados 724 Pintores, barnizadores, empapeladores y asimilados 725 Personal de limpieza de fachadas de edificios y deshollinadores 729 Otros trabajadores de acabado de construcción y asimilados 7N TRABAJADORES CUALIFICADOS DE LAS INDUSTRIAS EXTRACTIVAS, DE LA METALURGIA, LA CONSTRUCCIÓN DE MAQUINARIA Y ASIMILADOS 73 ENCARGADOS EN LA METALURGIA Y JEFES DE TALLERES MECÁNICOS 731 Jefes de taller y encargados de moldeadores, soldadores montadores de estructuras metálicas y afines 732 Jefes de taller de vehículos de motor 733 Jefes de taller de máquinas agrícolas e industriales y motores de avión 734 Jefes de equipo de mecánicos y ajustadores de equipos eléctricos y electrónicos 74 TRABAJADORES DE LAS INDUSTRIAS EXTRACTIVAS 741 Encargados y capataces de la minería 742 Mineros, canteros, pegadores y librantes de la piedra 75 SOLDADORES, CHAPISTAS, MONTADORES DE ESTRUCTURAS METALICAS, HERREROS, ELABORADORES DE HERRAMIENTAS Y ASIMILADOS 751 Moldeadores, soldadores, chapistas, montadores de estructuras metálicas y trabajadores asimilados 752 Herreros, elaboradores de herramientas y asimilados 76 MECÁNICOS Y AJUSTADORES DE MAQUINARIA Y EQUIPOS ELÉCTRICOS Y ELECTRÓNICOS 761 Mecánicos y ajustadores de maquinaria 762 Mecánicos y ajustadores de equipos eléctricos y electrónicos 7P TRABAJADORES CUALIFICADOS DE INDUSTRIAS DE ARTES GRÁFICAS, TEXTIL Y DE LA CONFECCIÓN, DE LA ELABORACIÓN DE ALIMENTOS, EBANISTAS, ARTESANOS Y OTROS ASIMILADOS Appendix 236 77 MECÁNICOS DE PRECISIÓN EN METALES, TRABAJADORES DE ARTES GRÁFICAS, CERAMISTAS, VIDRIEROS Y ARTESANOS DE LA MADERA, TEXTIL Y DEL CUERO 771 Mecánicos de precisión en metales y materiales similares 772 Trabajadores de artes gráficas y asimilados 773 Ceramistas, vidrieros y asimilados 774 Artesanos de la madera, de textiles, del cuero y materiales similares 78 TRABAJADORES DE LA INDUSTRIA DE LA ALIMENTACIÓN, BEBIDAS Y TABACO 780 Trabajadores de la industria de la alimentación, bebidas y tabaco 79 TRABAJADORES QUE TRATAN LA MADERA, EBANISTAS, TRABAJADORES DE LA INDUSTRIA TEXTIL, CONFECCIÓN PIEL, CUERO, CALZADO Y ASIMILADOS 791 Trabajadores que tratan la madera y asimilados 792 Ebanistas y trabajadores asimilados 793 Trabajadores de la industria textil, la confección y asimilados 794 Trabajadores de la industria de la piel, del cuero y del calzado 8 OPERADORES DE INSTALACIONES Y MAQUINARIA, Y MONTADORES 8Q OPERADORES DE INSTALACIONES INDUSTRIALES, DE MAQUINARIA FIJA; MONTADORES Y ENSAMBLADORES 80 JEFES DE EQUIPO Y ENCARGADOS EN INSTALACIONES INDUSTRIALES FIJAS 801 Encargados en instalaciones mineras 802 Encargados en instalaciones de procesamiento de metales 803 Encargados de taller de vidriería, cerámica y asimilados 804 Encargados de taller de madera y jefes de equipo en la fabricación de papel 805 Jefes de equipo en instalaciones de tratamiento químico 806 Jefes de equipo en instalaciones de producción de energía y asimilados 807 Jefes de equipo de operadores de robots industriales 81 OPERADORES DE INSTALACIONES INDUSTRIALES FIJAS Y ASIMILADOS 811 Operadores en instalaciones de la extracción y explotación de minerales 812 Operadores en instalaciones para la obtención y transformación de metales 813 Operadores en instalaciones para la obtención, transformación y manipulado del vidrio y la cerámica y asimilados 814 Operadores en instalaciones para el trabajo de la madera y la fabricación de papel 815 Operadores en plantas industriales químicas 816 Operadores en plantas para producción de energía y similares 817 Operadores de robots industriales 82 ENCARGADO DE OPERADORES DE MÁQUINAS FIJAS Appendix 237 821 Encargado de operadores de máquinas para trabajar metales 822 Encargado de operadores de máquinas para fabricar productos químicos 823 Encargado de operadores de máquinas para fabricar productos de caucho y de material plástico 824 Encargado de operadores de máquinas para fabricar productos de madera 825 Jefes de taller de imprenta, encuadernación y fabricación de productos de papel 826 Encargado de operadores de máquinas para fabricar productos textiles y artículos de piel y cuero 827 Encargado de operadores de máquinas para elaborar productos alimenticios, bebidas y tabaco 828 Encargado de montadores 83 OPERADORES DE MÁQUINAS FIJAS 831 Operadores de máquinas para trabajar metales y otros productos minerales 832 Operadores de máquinas para fabricar productos químicos 833 Operadores de máquinas para fabricar productos de caucho y plástico 834 Operadores de máquinas para fabricar productos de madera 835 Operadores de máquinas para imprimir, encuadernar y para fabricar productos de papel y cartón 836 Operadores de máquinas para fabricar productos textiles, artículos de piel y de cuero 837 Operadores de máquinas para elaborar productos alimenticios, bebidas y tabaco 84 MONTADORES Y ENSAMBLADORES 841 Montadores y ensambladores 849 Otros montadores y ensambladores 8R CONDUCTORES Y OPERADORES DE MAQUINARIA MÓVIL 85 MAQUINISTAS DE LOCOMOTORA, OPERADOR DE MAQUINARIA AGRÍCOLA Y DE EQUIPOS PESADOS MÓVILES, Y MARINEROS 851 Maquinistas de locomotoras y asimilados 852 Encargado de operadores de maquinaria de movimiento de tierras y de materiales 853 Operadores de maquinaria agrícola móvil 854 Operadores de otras máquinas móviles 855 Marineros de cubierta de barco y asimilados 86 Conductores de vehículos para el transporte urbano o por carretera 861 Taxistas y conductores de automóviles y furgonetas 862 Conductores de autobuses 863 Conductores de camiones 864 Conductores de motocicletas y ciclomotores 9 TRABAJADORES NO CUALIFICADOS 9S TRABAJADORES NO CUALIFICADOS EN SERVICIOS (EXCEPTO TRANSPORTES) 90 TRABAJADORES NO CUALIFICADOS EN EL COMERCIO 900 Vendedores ambulantes y asimilados Appendix 238 91 EMPLEADOS DOMÉSTICOS Y OTRO PERSONAL DE LIMPIEZA DE INTERIOR DE EDIFICIOS 911 Empleados del hogar 912 Personal de limpieza de oficinas, hoteles y otros trabajadores asimilados 92 CONSERJE DE EDIFICIOS, LIMPIACRISTALES Y VIGILANTES 921 Conserjes de edificios, limpiacristales y asimilados 922 Vigilantes, guardianes y asimilados 93 OTROS TRABAJADORES NO CUALIFICADOS EN OTROS SERVICIOS 931 Limpiabotas y otros trabajadores de oficios callejeros 932 Ordenanzas 933 Mozos de equipaje y asimilados 934 Lectores de contadores (agua…) y recolectores de dinero de máquinas expendedoras 935 Recogedores de basura y obreros asimilados 9T PEONES DE LA AGRICULTURA, PESCA, CONSTRUCCIÓN, INDUSTRIAS MANUFACTURERAS Y TRANSPORTES 94 PEONES AGROPECUARIOS Y DE LA PESCA 941 Peones agrícolas 942 Peones ganaderos 943 Peones agropecuarios 944 Peones forestales 945 Peones de la pesca 95 PEONES DE LA MINERÍA 950 Peones de la minería 96 PEONES DE LA CONSTRUCCIÓN 960 Peones de la construcción 97 PEONES DE LAS INDUSTRIAS MANUFACTURERAS 970 Peones de industrias manufactureras 98 PEONES DEL TRANSPORTE Y DESCARGADORES 980 Peones del transporte y descargadores