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1. Describe the properties of the following apical and basolateral amino acid transporters. a. Na+ dependent immino transporter (apical) Na+ uptake of Gly and Pro. Imino glycinuria disease. b. Cationic amino acid/cystine transporter (apical) Na+ independent transporter that couples uptake of cationic amino acids (ornithine, lysine, and Arg) Cystinuria. c. Na+ dependent neutral amino acid transporter (apical) Na+ dependent uptake of all neutral amino acids; activity with glycine and proline is limited. Hartnup disorder. d. Na+ dependent anionic amino acid transporter (apical) Na+ dependent transporter of Asp and Gln dicarboxylic aminoaciduria. e. Cationic amino acid transporters LAT1 and LAT2 (basolateral) What distinguishes the cationic amino acid transporters Lat1 and Lat 2 from each other? 2. Cystinuria a. What are the structural features of the transporter associated with this disease and how do mutations in the transporter result in Type A, Type B and type AB cystinuria? Two subunits held together by a disulfide bond. Type A has a mutation in the heavy chain. Homozygotes excrete large quantities of Cys, Lys, Arg, and Ornithine. Type B has a defect in the light chain Homozygotes excrete large quantities of Cys, Lys, Arg, and Ornithine. Heterozygotes are slightly effected and occasionally get cystine kidney stones. b. What are the clinical manifestations of cystinuria? Kidney stones. Excrete large quantities of cystine in the urine. Cystine is not reabsorbed in the kidney, low solubility is responsible for the pt. To have recurrent stones. Can cause acute or chronic renal failure due to obstruction. c. How is cystinuria treated clinically and what is the basis for the treatment? Adequate hydration, keeping urine dilute. Keep urine slightly alkaline to increase cystine solubility. Oral Kcitrate, NaCitrate, and NaCO3-. Reduction of dietary intake of cystine and met by reducing animal protein in diet. Treatment with agents that react with cystine to form more soluble disulfides with cysteine (thiopronin, penicillamine-cysteine dissulfide, penicillamine). 3. Hartnup Disorder a. What role do ACE2 and Collectrin have in regulating the transporter that is involved in this disease? Neutral amino acid transporter. Na+ dependent. Collectrin, a membrane protein involved in protein trafficking, stabilizes the protein and directs it to the cell surface. ACE2 stimulates the transport activity. Linked to poor Try uptake. In the kidney, the transporter foes not associate with collectrin and is not expressed at apical surface of proximal tubular epithelial cells. Neutral amino acids are not reabsorbed. Small intestine requires ACE2 for the expression of the transporter, unlike the kidney. b. What are the clinical manifestations of Hartnup disorder and how are they linked to the defect in amino acid transport? Rash with light sensitivity, psychosis, and cerebellar ataxia. Similar to pollegra. c. How is this disorder affected by diet? How is Hartnup disorder treated clinically and what is the basis for the treatment? Dietary niacin is treatment. Try plays a central role as a precursor to nicotinamide Nicotinamide comes mostly from diet but is also synthesized from Try, which is also a precursor to serotonin, likely explaining neurological manifestations. 4. Lysinurgic protein intolerance a. What are the properties of LAT1 and LAT2 and which transporter is involved in lysinurgic protein intolerance? LAT1 is involved in lysinurgic protein intolerance; LAT2 cannot pick up slack of LAT1 deficiency, is present in lower concentration.. Defective lys, arg, and ornithine uptake in small intestine and kidney, greatly increased excretion in urine and low level in plasma. Leads to derangement of the urea cycle due to ornithine levels. High levels of ammonia. b. What are the clinical manifestations of lyinurgic protein intolerance and how are they linked to the defect in amino acid transport? Develop an aversion to protein. Symptom free while breast fed. Failure to thrive as an infant. Postprandial (after eating)vomiting and diarrhea. Growth retardation, enlarged liver and spleen, muscular hypotonia, and osteoporosis. Episodic hyperammonemia. Various other renal and pulmonary complications. c. How is this disorder affected by diet? How is it treated clinically and what is the basis for the treatment? Treatment by promoting the excretion of nitrogen (NaBenzoate and phhenylbutarate), oral citrulline (precursor to ornithine and arginine, providing ornithine to promote urea synthesis to alleviate elevated blood ammonia), and place on a restricted diet that limits protein intake. 5. What is anion gap? How is it calculated? With potassium: It is calculated by subtracting the serum concentration of cholride and bicarbonate (anions) from the concentrations of sodium plus potassium (cations) = ([Na+]+[K+]) - ([Cl-] + [HCO3-]) Without potassium: Potassium is often ignored because its concentration, being low, usually has little effect on the calculated gap. Use the above equation, but set [K+] to 0. Anion gap reflects the anions that are not measured, e.g. proteins like albumin. In organic acidurias, organic anions contribute to anion gap. In an organic acidosis, the lost bicarbonate is replaced by the acid anion which is not normally measure d and the AG is increased. Defects in metabolism of branched chain amino acids Clinical presentations can include the following: Infant born through normal delivery is initially symptom free becomes restless and deteriorates with no obvious cause; onset may be 2 hours to two weeks following delivery, various neurological signs, poor feeding and drowsiness, progressive coma, respiratory distress, hiccups, apnea, bradycardia, and hypothermia. Disorders are differentiated based on profile of metabolites in serum, urine, and CSF and ultimately be assaying enzyme activity in cultured fibroblasts. Treatment is often specific, but all utilized diet limiting intake of offending amino acid. AA → aketo acids (by transaminase) → coa derivatives (by branched chain keto acid dehydrogenase complex) 6. Maple Syrup Urine Disease (MSUD) a. What enzyme complex is involved in this disease and what are its components? What reaction does this complex catalyze? Branched chain keto acid dehydrogenase. Three components can be affected: E1-keto acid decarboxylase (thiamine dependent) either alpha (IA) or beta (IB); E2-dihydrolipoyl acyltransferase (type II); E3 dihydrolipoamide dehydrogenase (type III). b. What are the clinical manifestations of this disorder? Encephalopathy 4-7 days after birth, branched chain amino acids, especially Leu, are elevated in blood;alloisoleucine is dignostic; lethargy and no interest in feeding; weight loss and progressive neurological deterioration; alternate hypotonia and hypertonia; characterisitic burnt sugar or maple odor to the urine; BCKD activity is less than 2% of normal. Intermediate MSUD: symptoms not as severe, branched chain amino acids persistently elevated, there is neurological involvement, BCKD activity is 3% to 30% of normal. Intermittent MSUD: normal early development and intelligence, symptoms usually appear between 5 mo and 2 years, stress (e.g. infection) can trigger acute metabolic decompensation. Thiamin responsive MSUD: Symptoms similar to intermediate form, alloisoleucine present in serum, treated with a low protein diet, administration of thiamine, along with diet, helps maintain low concentrations of BCAAs. c. What pattern of metabolites is seen in the serum and urine of patients with this disease? d. How is MSUD managed clinically and what is the basis for the treatment? Removal of toxic metabolites, continuous blood exchange transfusion or hemodialysis, should be initiated within hours of birth to prevent brain damage, leucine is partcularly toxic, thiamine may improve tolerance of BCAA in some patients (thiamine responsive types), patients generally healthy between episode between episodes of metabolic imbalance. Degree of mental impairment dependents on the length of time following birth when plasma leucine levels are elevated and the effectiveness of long term metabolic control. Most have some degree of mental impairment. 7. Isovaleric academia (IVA) a. What enzyme is involved in this disorder and what chemical reaction does it catalyze? Deficiency of Isovaleryl-CoA dehydrogenase (IVA), a mitochondrial flavin-dependent enzyme. Transfers electrons to electron-transfer flavoprotein (ETF), ultimately to reduce NAD. Catalyzes Isovaleryl-CoA → beta Methylcrotonyl-CoA. b. What is the prosthetic group of this enzyme? Flavin c. What are the clinical manifestations of this disease? First type: symptoms seen within 2 weeks of birth: vomiting, dehydration, listlessness, and acidosis. Second type: chronic and intermittent form, characterized by vomitting, lethargy, progressing to coma, elevated anion gap metablic acidosis, and ketonuria with a distinctive dirty sock smell, exacerbated in the first 12 months by various types of stress, such as infections, or ingestion of high protein foods. d. How is IVA managed clinically and what is the basis for the treatment? Dietary restriction (protein restriction) to limit intake of leucine, oral glycine and intravenous carnitine during episodes of metabolic crisis. 8. Methylmalonic Acidemia (MMA) and Propionic Acidemia (PA) a. What enzymes are involved in these diseases, what chemical reactions do they catalyze and what prosthetic groups are associated with these enzymes? Propionyl-Coa carboxylase, methylmalonyl CoA mutase. Propionyl CoA → methylmalonyl CoA → Succinyl-CoA. Biotin enzyme (PCC). Adenosylcobalamin enzyme (MCM) b. What are the clinical manifestations of MMA and PA? Accumulation of toxic metabolites, impairment of mitochondrial energy production, poor suckling or refusal to feed, vomiting, weight loss, abdominal distention, abnormal posturing and movemnts, generalized hypotonia, lethargy, serizures, appear shortly after birth with rapid deterioration for no apparent reason, patients can go into coma with brain edema, respiratory distress, hypothermia, and death within a few days. Lab findings: metabolic acidosis, increased anion gap due to accumulation of organic acids, ketonuria, anemia, hyperuricemia, leukopenia, often mistaken for sepsis, hyperammonemia is almost always present. Renal failure in MMA, Cardiomyopathy in PA related to poor uptake of carnitine, accumulation of toxic metabolites leading to inhibition of the TCA cycle and mitochondrial respiration in the brain, infarction of the globus pallidus with necrosis, spongiosis, and cyst formation that can normally be seen in an MRI. c. How are MMA and PA managed clinically and what is the basis for the treatment? Very little treatment, often involving managing metabolic crisis, but poor prognosis. 9. Nonketotoic Hyperglycinemia a. What enzyme system is involved in this disease and what are the components of this enzyme system? Glycine synthase has 4 proteins: P-protein (pyridoxal phosphate dependent decarboxylase), H-protein (small protein containing lipoamide covalently bound through a lysyl residue), Tprotein (an aminoethyl transferase), L-protein (FAD dependent lipoamide dehydrogenase). b. How is elevated glycine thought to be linked developmental problems and the central nervous system defects seen in patients with this disease? Glycine receptors in inhibitory neurons in the spinal cord may affect skeletal muscles in response to elevated glycine; NMDA glutamate receptor of neurons has a glycine binding site on one of the subunits that can promote glutamate dependent calcium influx through the receptor, and a decreased supply of N5N10CH2THF which is the precursor to N5GH3THF which interferes with the remethylation of met, the synthesis of DNA precursors as a precursor to other reduced folates, and as a methyl group donor in the reaction catalyzed by thymidylate synthase. c. How is nonketotoic hyperglycinemia managed clinically and what is the basis for the treatment? Reduce the activity of NMDA Glu receptor through diazapam, ketamine, or dextromethrphan. Promote the excretion of glycine through NaBenzoate. Control siezures through phenobarbital. 10. Urea Cycle Disorders You should know what reaction is involved in each of the disorders listed below and the enzyme(s) involved. 1. Hyperammonemia type 1 CPI, treatment with arginine stimulates CPI. N-acetylglytmate synthase deficience, treatment with carbamoyl glutamate activates CPI. 2. Hyperammonemia type 2 Ornithine transcarbamylase deficiency, treatment with high carbohydrate, low protein diet and benzoic acid or phenylacetic acid to treat ammonia intoxification. Elevated blood ammonia, amino acids, orotic acids, and glutamine. 3. Argininosuccinate synthetase deficiency Argininosuccinate synthetase, treatment with arginine, stimulates citrulline excretion. Citrulline and ammonia is elevated in plasma, CSF, and urine. 4. Argininosuccinate lyase deficiency Argininosuccinate lyase, Argininosuccinate → fumerate + arginine. Argininosuccinate and ammonia in plasma, CSF, and urine, usually fatal in first two years. Treated with arginine. 5. Arginase deficiency Argininase, Arginine +H2O → Ornithine + Urea. Treatment of diet of essential amino acids, minus arginine. Low protein diet reduces plasma ammonia. b. How urea cycle disorders managed clinically and what are is the basis for the treatment?