Question

Please explain your reasoning for each answer! Thank you.

A significant number of children are born with muscle weakness and necrosis, hypoglycemia, elevated ammonia in the blood (hyperammonemia), fatigue and heart disorders. These children also may have elevated acyl-carnitine in their blood, muscle and liver. As a result, all newborns are tested for the condition that is the focus of this project. (Tip: think about glycolysis: what pathways are regulated together? What pathways are increased in rate together when energy (or other significant molecules) are low or high in concentration?)

(a) How can carnitine and acyl-carnitine levels be accurately determined in blood, urine or in a tissue biopsy? Give at least one laboratory method that is actually used for each molecule

(b) The central theme of this medical condition is on the role of carnitine. Why is acyl-carnitine increased in concentration when free carnitine is low in concentration in all parts of liver cells (although there is one location on which you should really concentrate!)?

(c) Based on your answer to part (b), please explain the presentation in the clinic. That is, what led to low glucose, high fatty acids (i.e. acyl-carnitine) in liver cell cytosol and elevated levels of ammonia when you consider the metabolic pathways that you know and specifically the β-oxidation (fatty acid oxidation), gluconeogenesis, and the urea cycle?

Answer 1

Both skeletal and heart muscle are highly dependent on energy supply. As energy demand of these muscle tissues varies by several orders of magnitude, energy metabolism has to be tightly regulated in order to meet varying energy requirement. Inherited neuromuscular disorders affect approx. one in 3,500 children. Patients suffering from metabolic myopathies, muscle pain, weakness.

a) For an accurate diagnosis, biochemical tests with glycogen quantitation and determination of amylo-1,6-glycosidase activity in a liver biopsy(tissue biopsy) specimen or erythrocytes are required. Mutation analysis of the AGL gene may be helpful in some cases and has been advocated as a screening test using a commonly available test kit. In our opinion, primary screening by mutation analysis is inappropriate. A molecular diagnostic scheme has been proposed to diagnose GSDIII. Most of the mutations are nonsense mutations caused by a nucleotide substitution or small insertion or deletion. Some important genotype-phenotype correlations have emerged. Parental diagnosis is possible at enzymatic and genetic level.

Therapy- Curative therapy is not available for debranching enzyme deficiency. In order to avoid fasting hypoglycaemia in infancy, dietary measures have been proposed. Frequently daytime high protein feeding and supplementation of uncooked corn starch before sleep proved to be effective in young patients with regard to metabolic control.

b) Fatty acids are metabolized in the mitochondrial matrix to acetyl-CoA via beta-oxidation. Fatty acids are the major source of energy in man, especially during fasting. Most tissues are able to oxidize fatty acids to co2 and water. In addition, liver has the capacity to synthesize ketone bodies (acetoacetate and 3-hydroxybutyrate from acetyl CoA), which serve as an important fuel for extrahepatic organs, especially brain, during catabolism.Long chain fatty acids cannot freely cross the inner mitochondrial membrane and are shuttled via the carnitine system; acyl-CoA molecules are first coupled to carnitine, catalysed by carnitine-palmitoyltransferase I(CPT I), and the acylcarnitine complex then crosses the highly impermeable inner mitochondrial membrane. Acetyl-CoA derived from beta-oxidation, amino acid or glucose metabolism is channelled into citric acid cycle.

c)In humans, biosynthesis takes place in liver and kidney. To fulfill its function, intracellular carnitine concentrations have to be rather high, especially in skeletal muscle cells. Therefore, active carnitine transport mechanisms from blood into the cell have to exist. Translocation is mediated by the carnitine transporter, a special protein which located in cell membranes. The active transport of carnitine into tissue takes place against a concentration gradient, permitting tissue carnitine concentrations to be 20-to-50-fold higher than plasma levels. Furthermore, this carnitine transporter reabsorbs all carnitine from primary urine into blood. In urine, increased amount of lactate, pyruvate and ketone bodies may be found. Dicarboxylic aciduria reflecting secondary inhibition of fatty acid oxidation may be observed.Muscle biopsy is an important diagnostic tool. Mitochondrial proliferation may also become evident using the succinate dehydrogenase and cytochrome oxidase stains.