Chapter 192

## Abstract

Abstract

1. Membrane transport of cationic amino acids lysine, arginine, and ornithine is abnormal in four disease entities: classic cystinuria; lysinuric protein intolerance (hyperdibasic aminoaciduria type 2, or familial protein intolerance); hyperdibasic aminoaciduria type 1; and isolated lysinuria (lysine malabsorption syndrome). Cystinuria, the most common of these, is dealt with in Chap. 191. About 100 patients with lysinuric protein intolerance (LPI) have been reported or are known to me. Almost half of them are from Finland, where the prevalence of this autosomal recessive disease is 1 in 60,000. Autosomal dominant hyperdibasic aminoaciduria type 1 has been described in 13 of 33 members in a French Canadian pedigree, and isolated lysinuria has been described in one Japanese patient.

Arginine and ornithine are intermediates in the urea cycle; lysine is an essential amino acid. In lysinuric protein intolerance (LPI) (MIM 222700), urinary excretion and clearance of all cationic amino acids, especially of lysine, are increased, and these amino acids are poorly absorbed from the intestine. Their plasma concentrations are low, and their body pools become depleted. The patients have periods of hyperammonemia caused by “functional” deficiency of ornithine, which provides the carbon skeleton of the urea cycle. Consequently, nausea and vomiting occur, and aversion to protein-rich food develops. The patients fail to thrive, and symptoms of protein malnutrition are further aggravated by lysine deficiency.

2. Patients with LPI are usually symptom-free when breast-fed but have vomiting and diarrhea after weaning. The appetite is poor, they fail to thrive, and if force-fed high-protein milk or formulas, they may go into coma. After infancy, they reject high-protein foods, grow poorly, and have enlarged liver and spleen, muscle hypotonia, and sparse hair. Osteoporosis is prominent, and fractures are not uncommon; bone age is delayed. The mental prognosis varies from normal development to moderate retardation; most patients are normal. Four patients have had psychotic periods. The final height in treated patients has been slightly subnormal or low-normal. Pregnancies are risky: Profound anemia develops, platelet count decreases, and severe hemorrhages during labor and a toxemic crisis have occurred, but the offspring are normal if not damaged by delivery-related complications. Acute exacerbations of hyperammonemia have not been a frequent problem in treated patients, but may have been the cause of the sudden death in one adult male after moderate alcohol ingestion. About two thirds of the patients have interstitial changes in chest radiographs. Some patients have developed acute or chronic respiratory insufficiency, which in a few has led to fatal pulmonary alveolar proteinosis and to multiple organ dysfunction syndrome. Patients present with fatigue, cough, dyspnea during exercise, fever, and, rarely, hemoptysis, and may also show signs of nephritis and renal insufficiency. One adult patient with pulmonary symptoms has been treated with high-dose prednisolone and is in remission over 6 years after the occurrence of the symptoms. In another patient, bronchoalveolar lavages have produced immediate relief during several subacute exacerbations.

3. In LPI, the concentrations of the cationic amino acids in plasma are subnormal or low-normal, and the amounts of glutamine, alanine, serine, proline, citrulline, and glycine are increased. Lysine is excreted in urine in massive excess, and arginine and ornithine in moderate excess. Daily urine contains a mean amount of 4.13 mmol lysine (range 1.02 to 7.00), 0.36 mmol arginine (0.08 to 0.69) and 0.11 mmol ornithine (0.09 to 0.13) per 1.73 m2 body surface area. The mean renal clearances are 25.7, 11.5, and 3.3 ml/min/1.73 m2, respectively; occasional values suggest net tubular secretion of lysine. Cystine excretion may be slightly increased. Blood ammonia and urinary orotic acid excretion are normal during fasting but are increased after protein meals. The serum urea level is low to normal, and lactate dehydrogenase, ferritin, and thyroid-binding globulin levels are elevated.

4. The transport abnormality is expressed in the kidney tubules, intestine, cultured fibroblasts, and probably in the hepatocytes, but not in mature erythrocytes. In vivo and in vitro studies of the handling of cationic amino acids in the intestine and kidney strongly suggest that the transport defect is localized at the basolateral (antiluminal) membrane of the epithelial cells. In vivo, plasma concentrations increase poorly after oral loading with the cationic amino acids, but also if lysine is given as a lysine-containing dipeptide. Dipeptides and other oligopeptides use a different transport mechanism not shared with that of free amino acids. The dipeptide thus crosses the luminal membrane normally, and is hydrolyzed to free amino acids in the cytoplasm of the enterocyte. An efflux defect at the basolateral membrane explains why the dipeptide-derived lysine is unable to enter the plasma compartment in LPI. Direct measurements and calculations of unidirectional fluxes of lysine in intestinal biopsy specimens have confirmed that the defect indeed localizes to the basolateral cell surface. Similar cellular localization of the defect in the kidney tubules is suggested by infusions of citrulline, which cause not only citrullinuria but also significant argininuria and ornithinuria. Because citrulline and the cationic amino acids do not share transport mechanisms in the tubules, part of the citrulline is converted to arginine and then to ornithine in the tubule cells during reabsorption. A basolateral transport defect prohibits antiluminal efflux of arginine and ornithine, which accumulate and escape through the luminal membrane into the urine. The genetic mutations in LPI and possibly in all cationic aminoacidurias apparently lead to kinetic abnormalities in the transport protein(s) of the cationic amino acids. This is suggested by the fact that increasing the tubular load of a single cationic amino acid by intravenous infusion increases its tubular reabsorption, but reabsorption remains subnormal even at high loads. he other cationic amino acids are able to compete for the same transport site(s) also in LPI, but an increase in the load of one cationic amino acid frequently leads to net secretion of the others.

The plasma membrane of cultured fibroblasts shows a defect in the trans-stimulated efflux of the cationic amino acids; i.e., their flux out of the cell is not stimulated by cationic amino acids present on the outside of the cell as efficiently as it is in the control fibroblasts. The percent of trans-stimulation of homoarginine efflux in the fibroblasts of the heterozygotes is midway between that of the patients and the control subjects.

5. The exact cause of hyperammonemia in LPI remains unknown. The enzymes of the urea cycle have normal activities in the liver, and the brisk excretion of orotic acid during hyperammonemia supports the view that N-acetylglutamate and carbamyl phosphate are formed in sufficient quantities. Low plasma concentrations of arginine and ornithine suggest that the malfunctioning of the cycle is caused by a deficiency of intramitochondrial ornithine. This hypothesis is supported by experiments in which hyperammonemia after protein or amino nitrogen loading is prevented by intravenous infusion of arginine or ornithine. Citrulline, a third urea cycle intermediate, also abolishes hyperammonemia if given orally, because, as a neutral amino acid, it is well-absorbed from the intestine. Ornithine deficiency in LPI has recently been questioned because cationic amino acids and their nonmetabolized analogues accumulate in higher-than-normal amounts in intestinal biopsy specimens and cultured fibroblasts from LPI patients in vitro and the concentrations of the cationic amino acids in liver biopsy samples are similar or higher in the patients when compared to these concentrations in the control subjects. If hyperammonemia is not due to simple deficiency of ornithine, it could be caused by inhibition of the urea cycle enzymes by the intracellularly accumulated lysine; by a coexisting defect in the mitochondrial ornithine transport necessary for the function of the urea cycle; or by actual deficiency of ornithine in the cytoplasm caused by abnormal pooling of the cationic amino acids into some cell organelle(s), most likely lysosomes. The latter two explanations imply that the transport defect is expressed also in the organelle(s).

6. Lysine is present in practically all proteins, including collagen. Lysine deficiency may cause many of the features of the disease that are not corrected by prevention of hyperammonemia, including enlargement of the liver and spleen, poor growth and delayed bone age, and osteoporosis. Oral lysine supplements are poorly tolerated by the patients because of their poor intestinal absorption. ∊-N-acetyl-L-lysine, but not homocitrulline, efficiently increases plasma concentration of lysine in the patients, but acetyllysine or other neutral lysine analogues have not been used for supplementation.

7. Recently, a 622-amino-acid retroviral receptor (murine leukemia viral receptor REC1) with 12 to 14 potential membrane-spanning domains has been cloned. The physiological role of the receptor was soon found to be that of a cationic amino acid transporter at the cell membrane; the protein was hence renamed MCAT-1, mouse cationic amino acid transporter-1. The functional characteristics of the transporter are similar to those of system y+, a widely expressed Na+-independent transport system for cationic amino acids. The human counterpart of the mouse REC1 gene, encoding the retroviral receptor-transport protein, has been assigned to chromosome 13q12-q14 and named ATRC1. MCAT-1 (and y+) activity is not expressed in rodent liver, but two other related cationic amino acid transport proteins, formed presumably as a result of alternative splicing—Tea (T cell early activation; expressed also in activated T and B lymphocytes) and mouse cationic amino acid transporter-2 (MCAT-2)—are probably responsible for the low-affinity transport of cationic amino acids that is characteristic of (mouse) liver. Studies addressing the ATRC1 gene as well as the Tea and MCAT-2 genes as candidate genes for LPI are under way.

8. Treatment in lysinuric protein intolerance consists of protein restriction and supplementation with oral citrulline, 3 to 8 g daily during meals. Patients are encouraged to increase their protein intake modestly during citrulline supplementation, but aversion to protein in most patients effectively inhibits them from accepting more than the minimal requirement. The treatment clearly improves the growth and well-being of the patients. Pulmonary complications (interstitial pneumonia, pulmonary alveolar proteinosis, cholesterol granulomas, and respiratory insufficiency) have occasionally responded to early treatment with high-dose prednisolone, or to bronchoalveolar lavages. No therapy is known for the associated renal disease and renal failure.

9. The clinical and biochemical findings in other cationic aminoacidurias differ slightly from those in lysinuric protein intolerance. The symptoms of the index case with hyperdibasic aminoaciduria type 1 resemble those of LPI, but the other affected members of the pedigree are clinically healthy. The Japanese patient with isolated lysinuria has severe growth failure, seizures, and mental retardation. Her transport defect is apparently limited to lysine, and hyperammonemia is not a feature of the disease.

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