Carbohydrate metabolism

Cellular carbohydrate (CHO) metabolism involves both catabolic (glycolysis, glycogenolysis) and anabolic processes (glycogenesis, gluconeogenesis). Carbohydrates, as monosaccharides (glucose, fructose, galactose), are absorbed in the intestine and then transported to the liver where excess glucose, galactose and fructose are converted to glucose-6-phosphate (G-6-PO₄). Depending upon energy needs G-6-PO₄ undergoes either catabolism to form energy or anabolism to form glycogen, the storage form of glucose, in liver and muscles. To produce energy G-6-PO₄ (derived from monosaccharides from dietary CHO or produced from glycogen degradation by glycogenolysis) is converted by a series of enzyme reactions in the glycolytic pathway to form pyruvate or lactic acid, then to acetyl-CoA, which is also produced from fatty acid oxidation and degradation of the carbon skeleton of glucogenic amino acids (Table 17.1). Acetyl-CoA enters the Krebs cycle, also known as the citric acid or tricarboxylic (TCA) cycle, within the mitochondria. Within the Krebs cycle acetyl-CoA, combined with oxaloacetate, undergoes cycles involving eight enzymes, in which reducing equivalents are produced which then enter the electron transfer chain for the production of energy as adenosine triphosphate (ATP). When cells do not require G-6-PO₄ for energy production it undergoes glycogenesis to be stored as glycogen until required to restore blood glucose levels. G-6-PO₄ can also be produced via pyruvate from protein catabolism of glucogenic amino acids (Table 17.1) or breakdown of glycerol from lipids (gluconeogenesis).

Fat metabolism

Dietary fat is present mainly as long chain triglycerides, comprising a glycerol backbone and fatty acids. Dietary fats, and lipids produced endogenously from acetyl-CoA, are initially hydrolysed by lipases into glycerol and free fatty acids. Glycerol is then oxidised to acetyl-CoA via pyruvate. Lipid transport is a complex process and is discussed in Chapter 19. Fatty acids enter the mitochondria via the carnitine transport cycle (medium chain fatty acids enter independently of carnitine) into the β-oxidation spiral in which fatty acids, via a series of enzymes, produce acetyl-CoA and electron carriers. Acetyl-CoA can enter the Krebs cycle or form ketone bodies in the liver. Electron carriers (FADH₂ and NAD) enter the electron transfer chain to produce ATP. Acetyl-CoA in excess of requirements for energy production via the Krebs cycle is converted via lipogenesis to stored lipids in adipocytes.

Protein metabolism

Dietary protein is broken down into 20 individual amino acids for absorption. Of these nine are essential or indispensible (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine) as they cannot be synthesised by the body. The other 11 (alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glutamine, glycine, proline, serine, tyrosine) are classified as non essential amino acids (NEAA), or dispensable, as they can be produced from the breakdown of essential amino acids (EAA). A few of the NEAA become conditionally essential in certain disorders or at times of stress. These include arginine, cystine, glutamine, glycine, proline and tyrosine and in such circumstances must be provided in the diet.

Amino acids enter the body’s nitrogen pool (Fig. 17.2) and are used to form tissue protein for growth and body tissue repair, or are catabolised by two processes. The amine group (nitrogen moiety) undergoes transamination, in which it is transferred to a keto acid producing a keto acid and an amino acid, and then deamination to produce ammonia which is detoxified via the urea cycle to produce urea, which is then excreted in urine. The carbon skeletons of amino acids (organic acids) are glucogenic, ketogenic or both (Table 17.1).

Glucogenic amino acids degrade

• directly to Krebs cycle intermediates
  
• via acetyl-CoA or acetoacetyl-CoA to oxaloactetate which is either used in the Krebs cycle or can be converted to phosphoenolpyruvate and used to produce G-6-PO₄ via gluconeogenesis
  
• to pyruvate which can either be used to produce G-6-PO₄ via gluconeogenesis or converted irreversibly to acetyl-CoA by the pyruvate dehydrogenase (PDH) complex. Acetyl-CoA can then enter the Krebs cycle

Ketogenic amino acids degrade to acetoacetate or acetyl-CoA and are used in fatty acid synthesis or ketone body production (Fig. 17.3).

Control and regulation of metabolism

To maintain homeostasis within cells metabolic pathways have to be finely balanced (Fig. 17.4). This is achieved by either

• flux through pathways, increasing or decreasing by intracellular self-regulation of reactions (feedback) by responding to concentrations of substrates or products, e.g. a decreased concentration of an essential product causes enzyme activity in the metabolic pathway to increase and produce more product from the substrate
  
• responding to signals from other cells, involving hormones and growth factors

Influence of hormone regulation

Hormones are released according to the metabolic status including whether in the fed or starved state and stress. Their regulation of metabolic pathways play a crucial role by

• altering the availability of substrates, e.g. sympathetic response during exercise increasing the mobilisation of fatty acids, insulin being released in response to high blood glucose levels and glucagon being released when glucose levels are low
  
• influencing enzyme activity, e.g. epinephrine, a fight and flight hormone, increases phosphofructokinase (PFK) activity, an important enzyme in glycolysis. In addition glucocorticoids and thyroid hormones also affect energy metabolism

Table 17.2 summarises the action of hormones on metabolic pathways.

Hormone	Anabolic/ catabolic	Stimulates	Inhibits	Blood levels
				Glucose	Fatty acids	Amino acids
Insulin released in response to increased blood glucose increased blood amino acids	Anabolic	Uptake of glucose by muscle and adipose tissue glycolysis glycogenesis lipogenesis Cellular uptake of amino acids protein synthesis	Gluconeogenesis Glycogenolysis Lipolysis Fatty Acid Oxidation Proteolysis Ketogenesis
Glucagon released in response to decreased blood glucose decreased blood amino acids	Catabolic	Glycogen breakdown and formation of glucose glycogenolysis gluconeogenesis Fat breakdown lipolysis fatty acid oxidation	Glycogenesis Lipogenesis Protein synthesis
Epinephrine released during exercise and stress (stimulation of sympathetic nervous system)	Catabolic	Stimulates glucagon and cortisol release Glycogen breakdown and formation of glucose glycogenolysis gluconeogenesis Fat breakdown Lipolysis	Insulin release
Cortisol Released due to physiological and emotional stress Supports effects of decreased insulin and increased glucagon secretion	Catabolic	Decreased glucose, fatty acid and amino acid uptake by all tissues except brain gluconeogenesis lipolysis
Growth hormone Released in response to hypoglycaemia increased plasma amino acid levels increased metabolism associated with growth, exercise	Anabolic	Muscle protein synthesis Mobilisation of free fatty acids lipolysis Reduced cellular glucose uptake gluconeogenesis	Glucose uptake by muscles (glucose sparing)
Thyroxine Slow response to metabolic changes Increases sensitivity to epinephrine, glucagon and growth hormone Increases basal metabolic rate	Catabolic	Increases ATP production glycolysis glycogenolysis lipolysis fatty acid oxidation		indirectly

Inherited metabolic disorders

Inherited metabolic disorders (IMD) are genetically inherited biochemical disorders of specific enzymes or proteins causing a block in a normal metabolic process of protein, CHO or fat metabolism. As a result there is an abnormality in the normal metabolic process and accumulation of intermediary metabolites which cannot be further metabolised. This concept of IMD was originally conceived by Garrod [1] in 1902 and in 1908 he went on to describe such disorders including alkaptonuria, albinism, pentosuria and cystinuria [2] which are all inherited in an autosomal recessive trait. IMD can be categorised in a number of ways. Based on their pathophysiology they can be considered in three distinct groups.

1. Disorders of intermediary metabolism leading to intoxication
   

   These include disorders in which the enzyme deficiency leads to accumulation of intermediary metabolites proximal to the block (B) and the formation of alternative products (D) producing acute or chronic toxic effects which result in disease manifestations and may also lead to deficiencies of products downstream of the block (C).

   

   
   

   Examples of such disorders include the amino acidopathies (phenylketonuria, maple syrup urine disease, homocystinuria, tyrosinaemias); organic acidaemias; urea cycle disorders; disorders of sugar intolerance (galactosaemia, hereditary fructose intolerance); metal intoxication disorders (Menkes disease, Wilson disease) and porphyrias. These disorders all have a symptom free period after birth and then present with acute (either in the neonatal period or a later onset) or chronic signs of ‘intoxication’. Acute symptoms can be triggered by dietary intake, catabolism, fever, intercurrent illness. Chronic, progressive presentations include developmental delay, failure to thrive, cardiomyopathy, hepatomegaly, lens dislocation.

   
   
2. Disorders of intermediary metabolism involving energy metabolism
   

   These include disorders in which the enzyme deficiency results in lack of energy production or energy utilisation within organs including liver, muscle, brain, myocardium. These disorders can be subdivided into defects of mitochondrial or cytoplasmic energy:

   
   
   
   
   • mitochondrial energy disorders include disorders of fatty acid oxidation, ketone body defects, congenital lactic acidaemias, respiratory chain disorders
     
   • cytoplasmic energy disorders include disorders of glycolysis, glycogen metabolism, gluconeogenesis, glucose transport and hyperinsulinism and disorders of creatine metabolism
   
   

   Symptoms may include hypoglycaemia, lactic acidosis, hepatomegaly, cardiomyopathy, myopathy, hypertonia, failure to thrive, sudden infant death.

   
   
3. Disorders involving complex large molecules
   

   These are disorders in which complex molecules are incompletely catabolised and accumulate in organelles resulting in varied symptoms including progressive loss of function of affected body systems, organomegaly and dysmorphic features. They include disorders of lipoprotein and cholesterol metabolism, lysosomal storage disorders, peroxisomal disorders, disorders of glycosylation, disorders of sterol synthesis and disorders of purine and pyrimidine metabolism.

Management

A significant number of IMD are treatable, often by therapeutic dietary intervention. The main forms of nutritional therapy in IMD are:

1. substrate reduction in which the metabolite which accumulates, due to the enzyme deficiency, is restricted (if it is an essential nutrient) or as far as possible removed from the diet. Such a treatment strategy is employed in managing
   
   
   
   • disorders of amino acid metabolism
     
   • disorders of galactose and fructose metabolism
     
   • lipoprotein disorders
     
   • disorders of fatty acid oxidation
     
   • some peroxisomal disorders, e.g. Refsum’s disease
   
   
2. provision of products normally produced downstream of the block and which, as a result of the block, are conditionally essential to prevent deficiency
   
   
   
   • tyrosine supplementation in phenylketonuria
     
   • arginine in urea cycle disorders
     
   • maintaining normoglycaemia by regular feeding and/or CHO supplements in
     
     
     
     • glycogen storage disorders
       
     • disorders of fatty acid oxidation
     
     
   • cholesterol in Smith–Lemli–Opitz syndrome
     
   • mannose in CDG 1b (phosphomannose isomerase deficiency)
   
   
3. stimulation of residual enzyme activity by use of pharmacological doses of cofactors, often vitamins
   
   
   
   • vitamin B₁₂ in methylmalonic acidaemia
     
   • pyridoxine (vitamin B₆) in homocystinuria
     
   • riboflavin (vitamin B₂) in multiple acyl-CoA dehydrogenase deficiency
     
   • sapropterin in some forms of biopterin responsive phenylketonuria
   
   
4. providing alternative substrates
   
   
   
   • use of glucose and medium chain triglycerides in long chain fat oxidation disorders
     
   • use of the ketogenic diet in Glut 1 deficiency syndrome to produce ketones as an alternative energy source to glucose by the brain

In many IMD the promotion of anabolism and avoidance of catabolism is vital to prevent metabolic decompensation occurring. Where acute metabolic decompensation occurs with the risk of further irreversible damage, particularly neurological, the use of emergency dietary regimens is imperative. Detailed discussion of dietary treatment approaches are given in the relevant sections of this and the following chapters.

Patients with IMD are invariably complex to treat and it is important they are managed by an experienced metabolic team including doctors, specialist dietitians, clinical nurse specialists, biochemists, pharmacists and psychologists in a metabolic centre. Local professionals and hospital teams also form an integral part of the care for metabolic patients.

Newborn screening

Early detection is important to ensure a good outcome in many IMD, starting treatment before significant or any damage is done. Newborn screening (NBS) aims to detect affected infants before symptoms occur to allow early implementation of treatment. Phenylketonuria was the first IMD to be part of NBS (from the end of the 1960s); screening has been expanded to include a variety of other disorders, depending on local health policy. Further information is given in disorder specific sections.

Inheritance

The concept of IMD being due to a block in a metabolic process, with the accumulation of intermediary metabolites leading to disease, was introduced by Garrod [1, 2]. In 1911 Johannsen [3] introduced the theory that genes were responsible for inheritance and in 1941 Beadle and Tatum [4] hypothesised the one gene, one enzyme theory. The molecular disease concept in which gene mutations alter protein structure by changing a single amino acid within the protein was introduced by Pauling et al. [5] and Ingram [6] with their work on sickle cell anaemia.

The structure of DNA was described by Watson and Crick in 1953. DNA contains information as a code made up of four different chemical bases. The sequence of bases provides information for building and maintaining body structures. DNA sequences are made up of exons and introns. Exons are coding sequences which contain information to encode for a protein. Introns, which occur between exons, do not code for proteins. The process of a gene expressing for a protein involves two significant steps, transcription and translation. Transcription, within the cell nucleus, involves the transfer of information in the DNA to messenger RNA (mRNA). mRNA carries this information out of the nucleus into the cytoplasm for translation where ribosomes read the sequence of mRNA bases. Three base (codon) sequences code for specific amino acids (AA). Transfer RNA (tRNA) then assembles the protein by sequentially adding AA until a stop codon (a three base sequence which does not code for an AA) is reached [7].

Genetic disorders arise due to mutations (change) in the DNA sequence. An error in the coding sequence of a gene can result in no protein or an abnormal protein or enzyme being produced. Many mutations are benign (silent) whereas others lead to variable severity of defect. Mutation types include point mutations, missense mutations, nonsense mutations, deletions, insertions, inversions, duplication, frameshift mutations (Table 17.3). Further information can be obtained at www.genetichealth.com and www.ghr.nlm.nih.gov/handbook/mutationsanddisorders/possiblemutations.

Gene mutations result in a genotype which predicts residual enzyme activity. Single genes do not function independently but interact with other genes and the individual’s environment resulting in the disease phenotype. There is not always an exact genotype–phenotype correlation.

Patterns of inheritance

Autosomal recessive (AR) inheritance

In AR inheritance faulty (mutated) copies of the gene have to be inherited for the disease to be expressed (Fig. 17.5). Parents are obligate heterozygotes (carriers) but do not have the disease. They may carry the same or a different gene fault (mutation). Affected offspring are either homozygous with two copies of the same gene fault (mutation) or compound heterozygotes with two different gene faults (mutations). There is a 25% (one in four) risk of having an affected child with each pregnancy. Unaffected children have a 66% (two out of three) chance of being a carrier. Many IMD are inherited in this way including phenylketonuria (PKU), maple syrup urine disease (MSUD), galactosaemia, glycogen storage disease (GSD) Ia, medium chain acyl CoA dehydrogenase deficiency (MCADD).

When individuals affected with an AR disorder have children they will all inherit the faulty (mutated) copy of the gene. The risk of their children actually having the same AR disorder depends upon whether their partner is affected with the same AR disorder or carries a faulty (mutated) copy of the same gene. Children of parents who both have the same AR disorder will all inherit the disorder. The risk of offspring inheriting the disorder when one parent has an AR disorder depends upon the carrier frequency rate for the disorder. For example PKU has a carrier frequency of approximately 1 in 50 which gives a 1 in 100 (1%) chance of any child of a parent with PKU inheriting the condition.

Autosomal dominant (AD) inheritance

AD inheritance only requires one copy of a faulty (mutated) gene to inherit the disorder; this can be inherited from either parent (Fig. 17.6). There is a 50% risk of occurrence with each pregnancy. Unaffected offspring will have two normal copies of the gene. Dominant mutations can occur sporadically (de novo) with both parents having no mutations. This is often seen in Glut 1 deficiency syndrome. Occasionally an AD disorder can occur as the result of a germ line mutation; parents usually have a normal phenotype but one of the parents has a mutation occur in some of his/her sperm/eggs. AD conditions can occur in a homozygous state in which case they can be very severe or lethal. Familial hypercholesterolaemia (FH) is an autosomal dominant disorder; heterozygotes are symptomatic but homozygotes are more severe, often dying from fatal myocardial infarction in early adult life. Individuals with heterozygous AD disorders (only one copy of the faulty gene) have a 50% chance of passing on the faulty gene. All children to individuals with homozygous AD disorders will inherit the disorder.

X-linked inheritance

Mutations occur in genes on the X chromosome (Fig. 17.7). X-linked recessive single gene defects affect males as they only have one X chromosome and if the gene fault/mutation is passed on from a carrier mother then they are affected. Females carry the gene fault/mutation on one of their two X chromosomes. Depending upon the degree of X inactivation (how many of the mutant X chromosomes are active) females can be symptomatic to a greater or lesser extent, e.g. in ornithine transcarbomylase (OTC) deficiency some carrier females present with hyperammonaemia. Males with X-linked disorders will pass on the faulty gene to all of their daughters; sons are unaffected. Examples of X-linked IMD include adreoleukodystrophy, OTC deficiency, X-linked GSD type IX.

Classification of PKU

There is a broad continuum of phenotypes in PKU ranging from severe to milder forms. The need for diet therapy and the amount of dietary phenylalanine tolerated gives an indication of severity [24]. PKU is generally classified by the severity of hyperphenylalaninaemia at diagnosis.

• Classical or severe PKU is usually characterised by blood phenylalanine concentrations >1200 µmol/L, with a phenylalanine tolerance no greater than 250 mg/day to maintain plasma phenylalanine concentrations <360 µmol/L [25].
  
• Moderate PKU is classified with diagnostic phenylalanine concentrations of 600–1200 µmol/L. Most patients with moderate/severe PKU tolerate between 200 and 700 mg/day phenylalanine whilst maintaining plasma phenylalanine concentrations <360 µmol/L.
  
• Patients with persistent hyperphenylalaninaemia, or mild PKU, present with plasma phenylalanine concentrations between 120 and 600 µmol/L on a normal diet. If phenylalanine concentrations are maintained within these limits without treatment, there is little evidence to suggest patients are at risk of intellectual, neurological or neuropsychological impairment [26]. Some centres, however, advocate treatment when phenylalanine concentrations are >360 µmol/L [25, 27, 28].

Biochemistry

Phenylalanine hydroxylase catalyses the hydroxylation of phenylalanine to tyrosine in the presence of the cofactor BH4 and several enzymes that serve to regenerate BH4, e.g. dihydropteridine reductase and pterin-4-α-carbinolamine [32] (Fig. 17.8).

Normally hydroxylation of phenylalanine contributes up to 50% of the tyrosine that is incorporated into tissue protein [33]. In PKU there is an inability to convert phenylalanine into tyrosine. Subsequently low to normal or reduced concentrations of tyrosine are common in untreated patients. In addition the phenylalanine : tyrosine ratio, normally around 1:1, is significantly increased. Tyrosine is essential for the synthesis of the catecholamine neurotransmitters such as dopamine and norepinephrine.

Genetics

PKU is caused by mutations in the gene encoding the phenylalanine hydroxylase enzyme (PAH). The gene is on the long arm of chromosome 12 in the band region of q22-q24.1 [34]. There are more than 800 disorder causing mutations in the PAH gene [35, 36]. Different mutations affect the activity of PAH dissimilarly ranging from little or no effect to complete suppression of the activity of the enzyme [24]. There is a good correlation between genotype and phenotype [37]. Consequently genotypes with more severe mutations will result in a moderate to classical phenotype with higher untreated blood phenylalanine concentrations. Mild mutations in PAH will result in a phenotypically less severe form of PKU. In those cases where a patient is heterozygous for two mutations of PAH (i.e. each copy of the gene has a different mutation) the milder mutation will predominate. Spain has an especially high prevalence of mild PKU [25].

There are a small number of patients with so called severe PKU mutations who have escaped intellectual disability despite high blood phenylalanine concentrations and poor dietary control. These patients appear to have near normal brain phenylalanine concentrations despite high blood phenylalanine levels [38].

Diagnosis

In the UK PKU is detected on routine neonatal screening by collecting blood spots from the heel onto filter paper between days 5 and 8 of life, which are then analysed by tandem mass spectrometry (MS/MS) [39] (see www.newbornscreening-bloodspot.org.uk for screening guidelines for PKU in the UK). Infants with a phenylalanine level >240 µmol/L should be referred to a designated PKU team and assessed within 24 hours of a positive screening result. A repeat blood sample to recheck phenylalanine and tyrosine measurements, together with blood for dihydropteridine reductase and blood samples for pterins (neopterin and biopterin) to diagnose tetrahydrobiopterin deficiencies, should be taken. A low phenylalanine diet should be commenced immediately providing blood phenylalanine concentrations are ≥400 µmol/L (although some centres commence treatment when blood phenylalanine concentrations are ≥600 µmol/L [27]). It is a UK standard that all infants have commenced dietary treatment by 14 days of age [39].

It is good practice to test any new sibling of a proband case with PKU at 48–72 hours and then repeat at the usual newborn screening time (days 5–8). In the UK DNA analysis is used mainly for prenatal testing only or to detect carrier status of an ‘at risk relative’ [38].

Treatment guidelines

Diet is the basis of treatment for patients with PKU with blood phenylalanine concentrations consistently ≥400 µmol/L. It consists of a limited and controlled amount of natural protein derived from food sources to provide essential phenylalanine requirements, with the majority of nutrient requirements being met by a phenylalanine free source of L-amino acids, usually supplemented with added micronutrients (a protein substitute). The overall aim of treatment should be to ‘achieve an optimal cognitive and psychological development and well being of the child’ [40]. Published guidelines have been unanimous in their requirement for neonatal screening and strict blood phenylalanine control in the first 10 years of life [20, 41–43], but there has been little agreement about the upper blood phenylalanine threshold to commence treatment, phenylalanine target concentrations for adolescents and adults, the need for long term treatment, frequency of blood phenylalanine monitoring and use of alternative treatments such as large neutral amino acids (LNAA) and sapropterin. Currently a group of health professionals, working with the European Society for PKU (ESPKU), are developing PKU guidelines which, it is hoped, will be adopted throughout Europe and beyond. Recommended blood phenylalanine concentrations are given in Table 17.6.

Normal physiological plasma phenylalanine concentration in infants, children and adolescents is approximately 60 µmol/L [8].

Recent meta-analysis of blood phenylalanine concentrations and intelligence quotient (IQ) [44] support the approach that treatment should be continued in adulthood. Furthermore, recent meta-analysis of data from reaction times studies suggested that upper phenylalanine concentrations should be 320 µmol/L for children aged 7–13 years; 570 µmol/L for adolescents aged 14–18 years [45].

Outcomes in early diet treated PKU

Other treatment options

A number of new treatment options, either as an adjunct therapy to a low phenylalanine diet or to replace diet, are available or in development.

Prevalence

MSUD has an estimated frequency of approximately 1 in 116 000 [165]. In the Mennonite populations of Pennsylvania, Kentucky, Indiana and Wisconsin, USA, it occurs in approximately 1 in 176 newborns [158]. The frequency is also high in the Ashkenzi Jewish population and in countries where consanguineous marriage is common [160].

Classification

MSUD is a genetically and phenotypically heterogeneous disorder of variable clinical severity from a severe classical form to a milder variant. Five clinical and biochemical phenotypes are known (Table 17.9).

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Apr 1, 2017 | Posted by admin in NUTRITION | Comments Off on Disorders of Amino Acid Metabolism, Organic Acidaemias and Urea Cycle Disorders

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Type and BCKAD (% of normal activity)

Age of onset