Chapter 143. Disorders of Amino Acid Transport Across Cell Membranes

Epithelial cells in such places as renal tubules and intestinal mucosa utilize several different transport systems that move amino acids through the luminal (apical) and the antiluminal (basolateral) membranes of the cell in functional cooperation, utilizing sodium-dependent symporters, proton-motive forces, and concentration gradients of other amino acids. Each system prefers groups of amino acids with certain physicochemical properties, but most individual amino acids can use more than one transporter. The transport activities have been classified in five main groups: (1) the “basic system” for cystine and the structurally related dibasic cationic amino acids lysine, arginine, and ornithine; (2) the “neutral system” for neutral amino acids; (3) the “acidic system” for glutamate and aspartate; (4) the “iminoglycine system” for proline, hydroxyproline, and glycine; and (5) the “beta-amino acid system.”

Within the basic system, cystine, lysine, arginine, and ornithine are transported from the intestinal or renal tubular lumen into the epithelial cells by a luminal transporter in exchange for neutral amino acids. The dibasic amino acids are then transported from the epithelial cell into the tissues by an antiluminal (basolateral) dibasic amino acid transporter in exchange for neutral amino acids and sodium. Both these transporters are heteromers of a heavy subunit (N-glycosylated type 2 membrane glycoprotein) and a light subunit (nonglycosylated polytopic membrane protein) linked by a disulfide bridge.

The neutral transporter system is expressed only at the luminal border of the epithelial cells. It transports alanine, asparagine, citrulline, glutamine, histidine, isoleucine, leucine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine into the epithelial cells (Fig. 143-1).1,2As the number of molecularly identified amino acid transporters increases, the nomenclature based on gene homology in solute carrier families (SLC) will improve the accuracy of the classification.1,3

The principal selective aminoacidurias, cystinuria, lysinuric protein intolerance, and Hartnup disorder are caused by defects of the luminal cystine/dibasic amino acid transporter (encoded by the genes SLC3A1 and SLC7A9), the antiluminal or basolateral dibasic amino acid transporter (encoded by SLC7A7), and the neutral amino acid transporter (encoded by SLC6A19), respectively. Other rare transport defects include autosomal dominant dibasic aminoaciduria type I, isolated lysinuria, isolated cystinuria, iminoglycinuria, and dicarboxylic aminoaciduria. Knock-out mouse models have been developed for several transporter defects and will produce essential information on this complex system.1,2

Inherited defects in amino acid transport at the cell membrane (Fig. 143-1) are expressed as selective renal aminoaciduria (ie, the concentration of the affected amino acids is high in the urine while it is normal or low in plasma), intestinal absorption of these amino acids is almost always impaired. Their symptoms result from excess of certain amino acids in the urine or lack of them in the tissues. Consequently, in cystinuria, renal stones are formed because of high urinary concentration of poorly soluble cystine. In lysinuric protein intolerance, the transporter defect for the dibasic cationic amino acids leads to poor intestinal absorption and urinary loss of arginine, ornithine, and, particularly, lysine. Lack of the urea cycle intermediates arginine and ornithine leads to hyperammonemia and protein intolerance. and insufficient supply of lysine affects patients’ growth and immune systems. The pellagra-like dermatitis and ataxia in Hartnup disorder are attributed to deficiency of tryptophan, the precursor of niacin synthesis (Fig. 143-1).1-3

Cystinuria (OMIM 220100; OMIM 600918) causes a lifelong risk of urolithiasis.4-9Its average incidence is 1:7000 but varies considerably between different populations. A defect of the high-affinity luminal transporter for cystine and dibasic amino acids in the epithelial cells in the jejunal mucosa and in the proximal renal tubulus leads to poor intestinal absorption and poor reabsorption of these amino acids in the kidney. Normally, 99% of the filtered cystine is reabsorbed, but homozygotes with cystinuria excrete 600 to 1400 mg of cystine per day into urine. If the intratubular cystine concentration exceeds the threshold of solubility, crystals and stones will form. Sufficient amounts of cystine and dibasic amino acids are apparently absorbed from the intestine via alternative mechanisms (eg, in oligopeptide form), since no signs of deficiency of these amino acids have been described.

With increasing knowledge of the genetics of cystinuria, its classification is changing from phenotypic to mutation-based. Cystinuria has been classified into two subtypes: type I is the pure autosomal recessive form of the disease that represents over 60% of the cases, and non-type 1 is inherited in a dominant mode with incomplete penetrance. As obligate heterozygote carriers, the parents of type I patients have normal urinary amino acid profile, while the cystine excretion of the parents of non-type 1 patients ranges from normal to clearly elevated.

Type I cystinuria is caused by at least 103 mutations in the SLC3A1 gene on chromosome 2p, encoding the heavy subunit of the amino acid transporter rBAT. Non-type I cystinuria is caused by at least 66 mutations in the SLC7A9 gene on chromosome 19q, encoding the light subunit of the transporter, b0,+AT.8 A patient may also have inherited a type 1 allele from one parent and a non-type I allele from the other. Most of the mutations have been detected only in single patients, and the distribution of the more frequent ones is associated with ethnic background.9-14 A new genetics-based classification has been suggested: type A for SLC3A1 homozygotes (corresponding type I cystinuria), type B for SLC7A9 homozygotes (most of which represent non-type I cystinuria), and type AB for the mixed type.9 While SLC3A1 and SLC7A9 mutations, including recently reported unbalanced genomic rearrangements, explain most of the cases, it is possible that other genes may be involved.3,15SLC3A1gene is also associated with two recessive contiguous gene-deletion syndromes with neurological involvement: hypotonia-cystinuria syndrome and 2p21 syndrome.16

Some patients never develop kidney stones, but others have recurrent symptoms from early childhood, with acute episodes of abdominal or lower-back pain, hematuria, pyuria, or spontaneous passing of stones, often in clusters between long asymptomatic periods.17 There is a clear gender and individual variety in the risk of stone formation: The incidence of onset and total number of stone events is higher in males than in females, and marked differences are also seen between siblings sharing the same mutations, indicating a role for other genetic or environmental factors.18

Cystine stones are usually radio-opaque and visible on ultrasonography. They are often located in the bladder. A positive urinary nitroprusside test and analysis of urinary amino acids lead to the diagnosis. Homozygotes with cystinuria excrete more than 0.1 mmol cystine/mmol creatinine (250 mg cystine/g creatinine) into the urine, but the excretion varies markedly. Plasma concentrations of cystine and the dibasic amino acids are normal or slightly decreased. Chemical analysis of the stones alone may be misleading, because mixed stones are not uncommon in cystinuria, and some stones may contain no cystine at all. Excessive hydration to dilute the urine and alkalinization to improve cystine solubility are the cornerstones of therapy. Adults should consume up to 4000 to 5000 ml fluid/24 h (1.75 to 2 l/m2every 24 hours), 500 ml of this before bedtime and, if possible, 500 ml during the night. Amounts of at least 1000 ml/m2every 24 hours up to 3000 ml have been suggested for children.7Because cystine is much more soluble in alkaline urine (500 mg/l at pH 7.5 vs. about 250 mg/l at pH 7.0), permanent alkalinization of urine is beneficial. The aim is to keep the urinary pH constantly between 7 and 8. Potassium citrate in 2 to 4 single doses of 3 to 10 mmol is started with the lowest dose and adjusted by regular determination of urinary pH. As sodium increases urinary cystine excretion, sodium bicarbonate (1.5 to 2 mmol/kg per day in four doses) is used only in severe renal insufficiency that does not allow the intake of extra potassium. Restriction of dietary animal protein to limit endogenous cystine synthesis from methionine may be helpful, and moderate sodium restriction also decreases cystine excretion.7,19,20

If the standard therapy fails to prevent new stones or to dissolve preexisting ones, a thiol derivative is added to decrease urinary free cystine concentration by forming water-soluble compounds and by cleaving the disulfide bond of cystine to more soluble cysteine. 7,19,20 D-penicillamine (2 g/1.73 m2 body surface area up to 3 g/day in three doses) is well tolerated by most patients but may cause hypersensitivity reactions, renal problems, or a variety of autoimmune syndromes. Pyridoxine supplementation is needed during penicillamine therapy. Mercaptopropionylglycine (tiopronin) has fewer adverse effects but has occasionally caused glomerulopathy or hyperlipidemia. The dose (15 to 20 mg/kg per day up to 1000 mg/day in three doses) is adjusted individually. Captopril is well tolerated but may not be as effective as the other thiol compounds.7,19,20 Percutaneous nephrolithotomy and extracorporeal shock-wave lithotripsy may not be effective in stone removal, because cystine stones are extremely hard; these treatments are more effective in children than in adults. New, minimally invasive urological techniques (eg, ureteroscopic lithotripsy, laser probes, and in situ litholysis via percutaneous nephrostomies21) minimize the need for open surgery for stone removal.Surgical procedures should always be combined with conservative preventive therapy.7,22 Recurrent urinary tract infections, urinary obstruction, and renal insufficiency are possible complications.

Regular follow-up is essential to support treatment compliance, to monitor renal function, and to detect developing stones early. Weekly home monitoring of urinary pH and specific gravity, together with regular measurement of free cystine concentration (with a target of < 100 umol/mmol creatinine),23determination of cystine crystal volume in morning urine, or direct assessment of urinary supersaturation24,25may prove helpful. Early detection of the disease by screening the family members is valuable. Homozygotes with type I cystinuria frequently develop stones during the first decade of life and should perhaps be treated prophylactically from an early age. Other subtypes may have a milder course.26

Metabolic Derangement and Pathophysiology

Hyperammonemia after protein ingestion and protective aversion to high-protein foods make lysinuric protein intolerance (LPI; OMIM 222700) resemble urea cycle enzyme deficiencies (see Chapter 145). Only 130 patients have been reported, with the highest incidence in Finland (1:60,000); there are also clusters of families in Italy and Japan, and sporadic cases on all continents.27 The transport of the dibasic cationic amino acids lysine, arginine, and ornithine is defective at the basolateral membranes of epithelial cells in the renal tubules and small intestine.28-31Limited intestinal absorption of these amino acids and massive urinary loss of especially lysine result in low plasma concentrations, while the concentrations of neutral amino acids in plasma are slightly increased. Glutamine and alanine concentrations are often more clearly elevated due to the malfunction of the urea cycle. It is unclear if the transport defect is expressed in any nonepithelial cells. In contrast to an earlier report,32recent data indicates that fibroblasts from LPI patients have normal cationic amino acid transport, probably via other transporter isoforms.33 Erythrocytes also possess intact cationic amino acid transport.34,35Putative heteromeric structures involving various subunit isoforms are thought to exist in several tissues.27A transport defect in hepatocytes has been postulated because of normal or elevated cationic amino acid concentrations in liver biopsy in LPI, and the possibility of abnormal cationic amino acid transport between various intracellular compartments has been speculated.36

Two alternate promoters participate in tissue-specific regulation of the expression of the affected transporter gene.37 Many pathogenetic mechanisms of the variable multiorgan involvement in LPI are still unknown.38,39The malfunction of urea cycle in LPI is best explained by “functional deficiency” of the intermediates arginine and ornithine in the hepatocytes.36 Based on a limited number of small studies, there are interesting controversial data on the role of arginine and nitric oxide production in the pathophysiology of LPI, possibly reflecting real individual differences. Deficiency of arginine, the rate-limiting precursor of nitric oxide synthesis, may result in persistently low nitric oxide concentrations potentially influencing vascular endothelial and immunologic functions.40-42A similar mechanism has been suggested that impairs the function of alveolar macrophages, contributing to the development of alveolar proteinosis, a potentially fatal complication of LPI.43However, other investigators have found evidence for increased nitric oxide synthesis, mediated, in theory, by increased intracellular arginine concentrations that may result from the transport defect.44

Deficiency of the essential amino acid lysine probably has a role in poor growth and in skeletal and immunologic manifestations of LPI. Occasional patients have severe carnitine deficiency45due to dietary origin: The principal dietary source of carnitine is red meat, consumed in very small amounts by most patients with LPI. Chronic lysine deficiency also limits endogenous carnitine biosynthesis, and low plasma levels of dibasic amino acids may diminish growth hormone secretion.39An interesting finding that may contribute to understanding the growth problems in LPI is that IGF1 expression is downregulated in the mouse model of LPI, leading to fetal growth retardation.46

Clinical Presentation

The full natural history of LPI remains to be characterized, as most of the oldest patients are still in their forties or fifties. Postprandial episodes of hyperammonemia, with refusal to eat, vomiting, stupor, and unconsciousness, usually begin when the infants start receiving formula or other foods with higher protein content than breast milk.47,48Tube feeding may be fatal. Strong aversion to high-protein foods with failure to thrive is evident by the age of 1 year, aggravating the protein malnutrition and amino acid deficiencies. Children present with growth failure, hepatosplenomegaly, muscular hypotonia, and occasional fractures. Neurological development is normal if severe or prolonged hyperammonemia has been avoided. Bone maturation is retarded, and there is often marked delay of puberty.49,50In adults, the clinical heterogeneity of LPI is obvious. Most affected adults are of moderately short stature, with abundant subcutaneous fat on a square-shaped trunk and thin extremities. They have marked hepatomegaly with or without splenomegaly. Two thirds have skeletal changes, eg, osteopenia.49 but pathological fractures seldom occur. Radiological signs of pulmonary fibrosis are common, but few patients suffer from symptomatic interstitial lung disease.51 Mental capacity depends on previous history of hyperammonemia. Most patients have mild normochromic or hypochromic anemia, leukopenia, and thrombocytopenia, and their reticulocyte count is often slightly elevated. High serum immunoglobulin-G concentrations and abnormalities in the distribution of lymphocyte subpopulations and in humoral immune responses52-54have been reported.

Diagnostic Tests

The diagnosis is based on the combination of increased urinary excretion and low plasma concentrations of the cationic amino acids, especially lysine. The concentrations of plasma lysine, arginine, and ornithine are usually less than 80 μmol/l, 40 μmol/l, and 30 μmol/l, respectively. If plasma amino acid concentrations are exceptionally low due to very limited protein intake, urinary cationic amino acid excretion may sometimes be within the reference range. Hyperammonemia and orotic aciduria typically develop after protein-rich meals. Unspecific but consistent findings include elevated serum lactate dehydrogenase activity and increased serum concentrations of ferritin, thyroid hormone binding globulin, and zinc. Diagnosis can be confirmed by mutation analysis.

Genetics

At least 43 different mutations have been reported in the SLC7A7 gene on chromosome 14q, encoding the light subunit of the dibasic amino acid transporter.27,55-57 All Finnish patients share the same founder mutation 1181-2A>T that causes a frame shift and a premature stop codon.55The phenotypic variability is wide among the genetically homogeneous Finnish patients and among homozygous patients with other mutations, and no genotype/phenotype correlation has been established. The mode of inheritance is autosomal recessive.

Treatment and Outcome

The principal aims of treatment are to prevent hyperammonemia and to provide sufficient protein and essential amino acids for normal metabolism and growth (approximately 100 mg/kg per day of L-citrulline in three to five doses with protein-containing meals).58 As a neutral amino acid, citrulline is readily absorbed and partially converted to arginine and ornithine, improving the function of the urea cycle. For patients with constantly highly elevated glutamine and glycine levels, sodium benzoate or sodium phenyl butyrate may help to diminish the nitrogen load There is marked interindividual variation in the protein tolerance, and infections, pregnancy, and lactation may alter it extensively. Frequent home monitoring of blood ammonia in small children and follow-up of urinary orotic acid excretion and amino acid profile are necessary for optimal therapy. Correction of lysine deficiency in LPI is complicated by its poor intestinal absorption and the resulting osmotic diarrhea. However, L-lysine-HCl (20 to 30 mg/kg per day in three doses) elevate plasma lysine concentration to low-normal range without gastrointestinal or other side effects. We currently supplement all our patients with lysine,59but it is still too early to evaluate its long-term effects. Carnitine supplementation is indicated in evident carnitine deficiency. Due to the restricted diet, the patients need supplementary calcium, vitamins, and trace elements. 60 We and others61have successfully given growth hormone therapy to children with LPI whose severe growth retardation has not responded to improved nutrition and metabolic control.

There is an individual report of the benefit of bisphosphonate treatment for osteoporosis in LPI.62 The rare hyperammonemic crisis is treated following the guidelines for urea cycle disorders.63 All protein- and nitrogen-containing substances should be removed from the diet and sufficient energy supplied as intravenous glucose. Intravenous infusion of ornithine, arginine, or citrulline, beginning with a priming dose of 0.5 to 1.0 mmol/kg in 5 to 10 min and continuing with 0.5 to 1.0 mmol/kg per hour, will rapidly clear hyperammonemia. Sodium benzoate and sodium phenyl butyrate may also be used to utilize alternate pathways of ammonia elimination.63

Varicella infections are usually severe and can be fatal, and the patients should be immunized and treated with acyclovir.64Other complications include systemic lupus erythematosus65–67; bone-marrow involvement with erythrophagocytosis; and interstitial pulmonary disease with alveolar proteinosis, in some cases progressing to fatal multiorgan failure.51,68-72The treatment with immunosuppression or intravenous immunoglobulin is still experimental.65,73In alveolar proteinosis, bronchoalveolar lavage and steroid therapy have shown some effect.51,68-72,74,75 Granulocyte-macrophage colony-stimulating-factor therapy has recently been suggested for idiopathic alveolar proteinosis. It was tried in one child with LPI but was discontinued because of side effects. The child later received a successful heart-lung transplantation, but alveolar proteinosis recurred in the transplanted lungs.76 Recent reports indicate that variable degree of renal dysfunction is common in LPI, with Fanconi-type tubular problems (mild proteinuria, glucosuria, phosphaturia, tubular acidosis, and microscopic hematuria) and slowly deteriorating glomerular filtration that has led to end-stage renal disease and renal transplantation in individual cases.77,78

A recent survey detected such problems in 30% of the Finnish patients.78Pregnancies of LPI patients have been complicated by toxemia, anemia, or bleeding during the delivery, but many have been completed successfully.79Although hyperammonemia and the associated mental retardation can be avoided with current therapy, several other complications of LPI may develop. The accumulating knowledge of severe multisystem manifestations of LPI is radically changing the previous concept that the disease is usually a fairly benign and easily treated condition.

In Hartnup disorder (MIM 234500), pellagra-like dermatitis and neurological symptoms associated with a characteristic pattern of hyperaminoaciduria can be present. These symptoms were first reported in several members of the Hartnup family in 1956.80Since then, newborn screening programs have picked up several mostly asymptomatic subjects with the diagnostic urinary amino acid profile (estimated incidence 1 in 20,000).81 Due to a renal transport defect of neutral monoamino-monocarboxylic amino acids, the patients excrete alanine, serine, threonine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, histidine and citrulline, and the monoamino-dicarboxylic amidesasparagine and glutamine into the urine in five- to twentyfold excess. Their stools contain increased amounts of the same free amino acids,82 while the plasma concentrations are decreased or low normal. Bacterial degradation of unabsorbed tryptophan in the intestine produces large amounts of indole compounds excreted in the urine.The autosomal recessive disorder is caused by at least 10 mutations in the gene encoding the neutral amino acid transporter SLC6A19 at the luminal border of the epithelial cells in the renal tubuli and intestinal epithelium.81,83-86Recent data suggest that defects in other amino acid transporters may also result in the Hartnup phenotype.3The phenotypic heterogeneity of Hartnup disorder is puzzling: The monogenic transport defect probably develops into symptomatic disease only in association with certain polygenic and/or environmental factors. The clinical manifestations resembling niacin deficiency probably reflect deficient production of nicotinamide, a tryptophan metabolite. It has also been suggested that bacterial degradation products of tryptophan may be toxic to the central nervous system.87 On a protein-rich diet, most patients remain asymptomatic, most likely due to sufficient amino acid supply in peptide form.87In the few patients who develop clinical symptoms, the skin lesions on light-exposed areas and neurological problems, including intermittent cerebellar ataxia, headache, muscle pain, and weakness,88,89usually appear in early childhood and often ameliorate with age. Exposure to sunlight, infection, poor nutrition, or stress may precipitate the symptoms. Growth and developmental outcome are generally normal. Maternal Hartnup disorder seems to be harmless to the fetus.90 The characteristic excess of neutral amino acids in the urine and reduced or low-normal concentrations in plasma confirm the diagnosis. The symptoms usually disappear rapidly with oral nicotinamide (50–300 mg/day). Early recognition of the condition in newborn screening programs permits adequate follow-up and treatment. Clinical and molecular aspects of amino acid transport defects are summarized in Table 143-1.