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Whether crystals typical of cystinosis appear within the corneal stroma of a donor button transplanted into the eye of a patient with nephropathic cystinosis has become controversial.1-3We described a patient who had bilateral penetrating keratoplasties and nephropathic cystinosis, and who—when examined some 6 weeks after transplant—exhibited crystals typical of cystinosis within his donor button.1The same patient examined elsewhere 10 months after transplantation was described as having a clear corneal graft.2We had the opportunity to examine another patient with nephropathic cystinosis who also underwent penetrating keratoplasty and again to document the recurrence of corneal crystal deposition within a donor button. Report of a Case. —A 19-year-old woman with juvenile nephropathic cystinosis underwent two renal allografts. Her systemic medications included prednisone and azathioprine. Ophthalmic evaluation documented characteristic crystals of cystinosis within the conjunctiva and cornea. She presented with presumed pupillary block glaucoma of the left eye.

To study radiation effect separate from the microcirculation, fibroblasts were cultured from four patients with radiation wounds. Cells could be grown from irradiated tissue near the ulcer and from control normal tissue, but no cells could be cultured from the ulcers. The ability of radiation-treated fibroblasts to attach to the substrate and form colonies was less than that of unirradiated cells. Irradiated skin fibroblasts from the four patients had significantly longer mean generation times than did control cells. During log-phase growth (1 to 9 days), the population doublings of damaged cells were significantly reduced compared to colonies from normal cells. These data suggest a permanent intrinsic radiation effect on fibroblasts or a selective ablation of faster-growing fibroblast subpopulations that is not dependent on decreased blood supply.

We treated 93 children with nephropathic cystinosis with oral cysteamine (mean dose, 51.3 mg per kilogram of body weight per day) for up to 73 months. This agent is known to be effective in depleting cells of cystine. In our study, the mean cystine depletion from leukocytes was 82 percent. A historical control group of 55 children received either ascorbic acid (27 children) or placebo (28). At age six, 2 of 17 controls had a serum creatinine level less than 1.0 mg per deciliter, as compared with 17 of 27 patients treated with cysteamine for at least one year (odds ratio, 12.8; 95 percent confidence interval, 2.1 to 33.9). At the end of the study, creatinine clearance was higher in the cysteamine group than in the control group (38.5 vs. 29.7 ml per minute per 1.73 m2; 95 percent confidence limits on the difference, 1.8 and 15.8), even though the cysteamine group was on average 1.4 years older than the control group. Cysteamine also improved growth; those in the cysteamine group between two and three years of age grew at 93 percent of the normal velocity, as compared with 54 percent in the control group. Fourteen percent of the patients could not tolerate the taste and smell of cysteamine. Concurrent controls treated in a blinded fashion with a placebo were not included in this study. With this limitation in mind, we conclude that oral cysteamine, by depleting cells of cystine, helps maintain renal glomerular function, improves growth, and constitutes the current treatment of choice for nephropathic cystinosis.

• Cystinosis is a rare autosomal recessive metabolic disorder in which nonprotein cystine accumulates within most body organs due to a defect in lysosomal cystine transport. The pathognomonic ocular manifestations of cystinosis are the presence of distinctive iridescent crystals within ocular tissue and a pigmentary retinopathy. We measured spatial contrast sensitivity in seven patients with infantile-onset nephropathic cystinosis and compared their contrast sensitivity function with that measured in ten age-matched controls. Spatial contrast sensitivities in the patient group were significantly lower than those in the normal group. Loss of contrast sensitivity in the patients with nephropathic cystinosis was more pronounced at higher spatial frequencies. We speculate that this loss of contrast function is primarily a manifestation of corneal disease, with secondary contributions from retinal changes and central nervous system dysfunction.

We studied 15 patients with infantile nephropathic cystinosis. We found that anterior corneal cystine crystal deposition began early in life and proceeded posteriorly as the patient aged; deposition advanced more rapidly in the periphery. Ultrastructural analysis of a corneal button obtained from a 20-year-old patient undergoing corneal transplantation confirmed our clinical observations that crystals were deposited throughout the entire central stroma.

Publisher Summary The cystine-binding protein is utilized for cystine transport through the osmotic shock-sensitive transport system of Escherichia coli. This protein is located in the periplasmic space and can be isolated following osmotic shock of this bacterium. It reversibly binds cystine with a dissociation constant of 10 n M . When purified, the cystine-binding protein may be used as described to measure cystine levels in physiological fluids. The chapter also discusses the use of cystine-binding protein for quantitative cystine assay in biological samples. Nanomolar concentrations of the disulfide, cystine, are assayed based on the competitive binding between [ 14 C] cystine and the nonradioactive cystine present in a sulfosalicylic acid extract of the biological material. The cystine-binding protein does not bind reduced glutathione or other thiols that are present in much larger amounts than cystine in most biological samples, eliminating the need for purification procedures prior to assay. The specificity of the cystine binding is discussed.

Summary: The free (nonprotein) cystine content of human cystinotic fibroblasts was found to vary with the pH of their culture medium. Intracellular cystine content was highest at alkaline medium pH's and lowest at acidic medium pH's. When maintained in medium of pH 6.3, cystinotic fibroblasts lost 50% of their free-cystuie content in 6 hr and over 95% in 30 hr. Cystine was the only amino acid to show significant variation with changes in the pH of the culture medium.

We diagnosed cystinosis in an 18-week-old fetus on the basis of an increased content of nonprotein cystine in cultured amniotic-fluid cells. These cells did not contain cystine crystals, a...

Cystinosis is a rare autosomal recessive metabolic disorder in which non-protein cystine accumulates within cellular lysosomes owing to a defect in lysosomal cystine transport. The pathognomonic ocular manifestation of cystinosis is the deposition of distinctive iridescent crystals in the cornea, not associated with any inflammatory response or recognised change in corneal function. We measured corneal thickness in nine patients with infantile nephropathic cystinosis. We also studied a corneal button from one of these patients who underwent corneal transplantation. All nine patients had increased corneal thickness in comparison with an age matched control population. Electron microscopy analysis of the cystinotic button revealed structural changes of both epithelium and endothelium layers. Increased corneal thickness in patients with nephropathic cystinosis may reflect subclinical corneal oedema.

Human skin fibroblasts derived from patients with nephropathic cystinosis were transformed with SV40 virions, cloned and permitted to enter the degenerative stage of growth termed "crisis," characteristic of SV40 transformed human cells. Nephropathic cystinosis is an autosomal recessively inherited metabolic disorder resulting in the intracellular accumulation of the amino acid cystine. A transformed cystinotic cell line which was recovered from the crisis stage was indistinguishable from its transformed precrisis parental cell strain in growth rate in media containing either 1% or 10% serum, cloning efficiency on plastic, in semisolid media, or upon confluent monolayers of normal skin fibroblasts, expression of SV40 T antigen, or production of virus. However, the modal DNA content of the recovered postcrisis transformed cystinotic cell line was different from that of the cloned parental precrisis transformed cell strain, suggesting that the postcrisis line was derived from a small subpopulation of the precrisis strain. The DNA content of the established cystinotic cell line continued to be unstable during subsequent subculturing and gave rise to subclones with both more and less DNA per cell. This line now has an apparently infinite growth potential and still has the hallmark of the cystinotic parental line, the storage of abnormally large amounts of intracellular nonprotein cystine.

A method was developed for the isolation of unmodified lysosomes of human origin using cultured EB-virus transformed lymphoblasts. The cells were lysed carefully by repeated resuspension in buffered isotonic sucrose. A crude granular fraction derived from this lysate was further purified by isopyknic centrifugation in an isotonic colloidal silica gel gradient and by free-flow electrophoresis. The following relative specific activities (mean ± S.D.) of lysosomal marker enzymes were measured in a pooled lysosomal fraction obtained from the final electrophoresis step (representing less than 0.1% of the initial protein): β-N-acetylglucosaminidase 85.6 ± 15.5; β-galactosidase 87.6 ± 13.4; acid β-glycerophosphatase 41.7 ± 3.5; β-glucuronidase 36.6 ± 6.1. With respect to the final two enzymes the recovery within this pooled fraction was 5–6% of the initial lysate. The great differences in relative specific activities achievable may be due mainly to different extralysosomal portions of the lysosomal marker enzymes, as was found for acid β-glycerophosphatase which was largely distributed within non-lysosomal structures in lymphoblasts when studied by histochemical staining. The final fraction consisted almost exclusively of lysosomes when examined by electron microscopy. Most lysosomes appeared club-shaped immediately after cell lysis and throughout the preparation procedure. Examination by electron microscopy and measurement of the latency of lysosomal enzyme activity revealed an exceptional integrity of the lysosomal membrane. This method provides the opportunity to study highly purified lysosomes from patients with lysosomal disorders.

We identified 80 patients with nephropathic cystinosis older than age 10 years in the United States and Canada. The oldest reported individual was 26 years of age. Ninety percent of patients had received at least one renal allograft. Age at the time of first transpiant varied between 7 and 17 years (mean 10.0 years). Almost three fourths of the patients required thyroid replacement, 27% had splenomegaly, and 42% had hepatomegaly. Photophobia was noted in 86% of patients, decreased visual acuity in 32%, and corneal ulcerations in 15%. Neurologic involvement, renal osteodystrophy, and diabetes mellitus were unusual. All these late complications of nephropathic cystinosis contribute to a description of the natural history of the disease and provide a rationale for the therapeutic use of cystine-depleting agents after renal transplantation.

Cystinosis is a recessively inherited disorder characterized by an accumulation of free cystine within lysosomes. The lysosome is a major site of intracellular protein degradation, which results in...

Free (non-protein) amino acids were measured in whole rat liver and in unmodified lysosomes which were prepared from rat liver by the technique of free-flow electrophoresis. Significant intralysosomal pools of threonine, serine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and arginine were found. No efflux occurred from rat liver lysosomes in isotonic buffered sucrose at 0°C, but all amino acids showed various degrees of efflux at 20 0 and 37 0 .

Tissue culture is a valuable tool for the study of cellular amino acid metabolism. However, accurate measurement of intracellular amino acids is difficult. Cells must be washed to remove the culture medium which is rich in amino acids, a procedure which may result in the loss of intracellular amino acids. Collection of the cells for washing and amino acid measurement is further complicated by the adherence of cultured fibroblasts to the culture vessel surface. An uncomplicated, reliable method for the determination of the amino acid content of cultured human diploid fibroblasts is described. The intracellular levels of several amino acids determined by this method are markedly higher than previously reported levels.

Certain aminothiols rapidly deplete cultured cystinotic skin fibroblasts of their abnormally high free (nonprotein) cystine pool. The free cystine content of these cells if reduced by over 90% in 1 h with 0.1 mM cysteamine. This is more rapid than previously known methods of removing free cystine from cystinotic fibroblasts. The disulfide, cystamine, is also able to deplete cystinotic cells of free cystine. A patient with nephropathic cystinosis and end-stage renal disease was treated with cysteamine, both intravenously and orally. Both methods of administration rapidly lowered the free cystine content of the patient's peripheral leukocytes. Study of the patient's urinary sulfur excretion did not conclusively determine the effect of this therapy on the total body cystine pool. Her renal status remained at end stage after 1 mo of oral cysteamine, when an episode of grand mal seizures prompted cessation of the study. Determination of the proper place of aminothiol therapy in this disease will depend upon further clinical trial with patients whose kidney function has not deteriorated to the point of irreversible change, accompanied by careful monitoring of plasma aminothiol levels.

We measured corneal sensitivity in 14 patients with infantile nephropathic cystinosis and in 13 age-matched controls. All patients with cystinosis had the pathognomonic anterior segment findings of crystal deposition within conjunctiva and cornea. Transcutaneous stimulation of the supraorbital nerve and surface electromyographic recording of the orbicularis oculi muscle performed on four patients showed a normal afferent limb to the blink reflex. The corneal sensitivity in patients with cystinosis was 3.18 g/mm2; in the control subjects it was 0.43 g/mm2. This difference was statistically significant (P less than .001).

Three unrelated adult patients with cystinosis were studied in an attempt to explain the differences in the clinical manifestations of childhood and adult cystinosis. The free cystine cont...

CHILDREN with cystinosis rarely live beyond 10 years of age. 1-3 Because of their early death and the rarity of this disease it is not surprising that there have been no previous reports (to our knowledge) of appendicitis occurring in children with cystinosis. The occurrence of both disease processes in the same patient presented an interesting diagnostic problem. Report of a Case The patient (NIH-06-33-53) was a 3½ -yearold white boy who had cystinosis. The diagnosis was made when he was 20 months of age on the basis of the following findings: (1) renal tubular defects consistent with the Fanconi syndrome, (2) demonstration of cystine crystals in bone marrow aspirates, (3) refractile bodies in the cornea and conjunctivitis seen by slit-lamp examination, and (4) retinopathy typical of cystinosis. 4 A more detailed clinical description may be found elsewhere. 4,5 For the previous 18 months his diet had consisted of a

The intracellular concentrations of reduced cysteine and glutathione are normal in cystinotic fibroblasts, even though they contain a marked increase in cystine. Cystine accumulation is thus not attributable to an elevated concentration of cysteine with secondary cystine formation, nor can it result from a decreased concentration of reduced glutathione, which acts as a hydrogen donor in transhydrogenation reactions for maintaining intracellular cyst(e)ine in its reduced state. The rates of turnover of glutathione in cystinotic and normal fibroblasts are similar, with half-lives of about 6–8 h. The intracellular cystine pool in cystinotic cells has a half-life of approximately 6 h, indicating that while compartmentalized, it remains in a state of dynamic equilibrium with exogenous cystine. The intracellular cysteine and glutathione levels in normal amniotic fluid cells are similar to those in fibroblasts. This suggests that the metabolism of these substances is similar in both types of cells, and strengthens earlier suggestions that cultured amniotic fluid cells may be used for the prenatal diagnosis of cystinosis, as fibroblasts are now used for its diagnosis postnatally.

In patients with cystinosis, the concentration of free cystine in leukocytes was 80 times greater than normal, and six times the normal content for their parents. This is the first demonstration of an abnormality in heterozygotes for this rare inherited disease of childhood. Three-quarters of the cystine was recovered in the granular fraction of cystinotic leukocytes.

The presence of a significantly increased content of free-cystine in skin fibroblasts from both homozygotes and heterozygotes for cystinosis emphasizes the central role of cystine in this disease, even though the primary defect responsible for cystine accumulation is yet to be determined. The studies described in this communication provide evidence that cystine is compartmentalized in a subcellular location in cystinotic cells. In fact, the very growth of cystinotic fibroblasts in the presence more than 100 times the usual content of free-cystine is evidence that the accumulated cystine is not freely dispersed throughout the cell, since would otherwise inhibit many enzymes requiring free sulfhydryl groups for activity (Patrick, 1965). We have no evidence as to whether the cystine is located in a known subcellular organelle or in a previously unrecognized location. Skin fibroblasts may provide a convenient tool to pursue these questions.

SINCE the discovery of somatic cell hybridization1 this technique has been used primarily for studies of formal genetics, cell differentiation, and cancer2. This study represents an attempt to use this technique to study a metabolic disease in which the primary defect is unknown.

Nephropathic cystinosis is an autosomal recessive disease characterized clinically by renal tubular and glomerular dysfunction and biochemically by intralysosomal cystine accumulation. We have recently reported that aminothiol treatment of cultured fibroblasts from patients with cystinosis produces rapid and complete removal of the stored cystine (1). Subsequent studies (2) showed that such cystine-depleted cells can reaccumulate cystine during incubation in culture medium lacking cystine. Thus, cystine appears to accumulate in these cells from an intracellular source. Potential intracellular sources include (1) GSH, (2)protein, and (3) cystathionine. No activity of the cystathionase pathway has been found by previous investigations in these cells, and none was found under the conditions of cystine depletion in the current investigations. Cells depleted of >80% of their GSH content reaccumulate cystine to the same extent as non-GSH depleted controls. Cells selectively labelled with 35S-cystine in either the GSH + protein pools or GSH pool alone only reaccumulate labelled cystine when the protein pool is labelled. Known inhibitors of protein degredation (NH4Cl and chloroquin) reversibly inhibit cystine reaccumulation in cystine-depleted cells. We conclude that the intralysosomal cystine accumulation observed in cells cultured from patients with cystinosis is derived from protein catabolism. (1) Thoene, J.G. et al. J.Clin.Invest.58:180, 1976. (2) Oshima, R.G. et al. J.Biol.Chem. 251:4287, 1976.

Defective cystine efflux has been demonstrated in lysosomes from both peripheral leukocytes and cultured EBV-lymphocytes from cystinotic patients. However, this abnormality has been difficult to demonstrate in fibroblast lysosomes, raising the question of whether lysosomal transport is the only defect in cystinosis. We have substantiated that some previous methods of loading fibroblast lysosomes with cystine, using cystine dimethyl ester (CDME), lead to cystine accumulation in a non-lysosomal as well as the lysosomal compartment (E. Harms, personal communication). If fibroblasts are removed from their anchorage substrate and incubated with 0.25 mM CDME for 15 min., only lysosomes are loaded with cystine as shown on Percoll gradients. In cystine-loaded lysosomes from two normal control fibroblast cultures the 20 min. cystine efflux was 19% and 20% without ATP and 56% and 74% with 2 mM MgATP. Lysosomes from two cystinotic fibroblast cultures had no cystine efflux over 20 min. both with and without ATP. We have used the dye, dipropylthiodicarbocyanine iodide to measure lysosomal membrane potential. MgATP causes a positive shift in membrane potential, in both cystinotic and normal lysosomes. In both cases, inhibitor and anion effects are consistent with an electrogenic proton pump. All of these findings support the concept of an abnormal lysosomal cystine transport protein as the primary defect in this disease.

Heterozygotes for the autosomal recessive disease cystlnosis are currently detected by measuring the cystine content of mixed leukocyte preparations. This method offers about 90% accuracy. The following study was designed to determine if measuring the cystine content of purified preparations of polymorphonuclear leukocytes (PMNs) would improve the accuracy of heterozygote detection. Subjects included 29 obligate heterozygotes for nephropathic cystinosis and 18 individuals presumed to be normal. Mixed leukozytes were prepared from 10 ml of blood by dextran sedimentation; PMNs were prepared by centrifugation of 4.5 ml of blood on a discontinuous gradient of Ficoll-Hypaque. The cystine content of both leukocyte preparations was determined by a specific binding assay. All values are expressed as nmol 1/2 cys/mg protein and are shown in the table below. Using mixed leukocyte preparations, 3 heterozygotes overlapped the normal range reconfirming a detection accuracy of 90%. Using PMNs no heterozygote values fell within the normal range. Measuring the cystine content of PMNs appears to provide a simple screening assay which improves the accuracy of heterozygote detection for cystinosis.

Objective:To review the history, basic defect, pathogenesis, clinical manifestations, diagnosis, and treatment of nephropathic cystinosis. Design:Lysosomal membrane transport studies, clin...

1136 URINARY EXCRETION OF GAMMA-HYDROXYBUTYRIC ACID IN A PATIENT WITH NEUROLOGICAL ABNORMALITIES; A NEW INBORN ERROR OF AMINO ACID METABOLISM?

I-cell (MLII) fibroblasts have a high free-cystine (cys) content. As in cystinotic (C) cells, the cys is in lysosomes. In cystine-free media with 1mM cysteamine, both cell types lose 50% of their cys in 6-9 min. The rate of loss changes with time, and by 60 min the decrease of cys is 88-97% in C cells but only 54-64% in MLII cells. Following removal of cysteamine and replacement with complete medium, cys reaccumulation in the first 8 h.is much greater in C cells (0.27-0.48 nmol/mg prot/h.) than in MLII cells (0.02-0.04 nmol/mg/h.). From 8 to 24 h. the rates of reaccumulation of both cell types are similar (0.06 nmol/mg/h.). Reaccumulation of cys in complete medium containing 30 mM glutathione-cysteine disulfide (GSSC) is increased 380% in C cells, but only 140% in MLII cells. With 50 μM chloroquine, the reaccumulation of cys in complete medium is inhibited in both cell types. Chloroquine does not inhibit the marked increase in cys reaccumulation with GSSC seen in C cells, but does in MLII cells. This suggests that the mechanism of cystine storage differs in the two cell types and that protein degradation is a more important source of cys in MLII than in C cells.

The cystine content of cystinotic fibroblasts increases with the length of time in cultivation. Four of five cystinotic cell strains showed an increase in cystine content with increased passage number. Cystinotic fibroblasts did not increase in cell number after 3 days in dialyzed fetal bovine serum. However, their cystine content increased almost 2-fold on a per cell number basis. Cystinotic cells incubated in cystine-free medium lost approximately 50% of their free cystine over a period of 12 hr and subsequently remained at about this level for 12 additional hr. Treatment of cystinotic cells with 1.0 mM dithiothreitol in complete medium reduced their free cystine content to about 25% of the initial level in 5 hr. The free cystine remained at this level after incubation overnight incystine-free medium without dithiothreitol. When dithiothreitol is added in the presence of a cystine-free medium, even more free cystine is removed from cystinotic cells (90%), and the free cystine content remains at this low level for as long as 48 hr if the cells are kept in a cystine-free medium. Cystinotic cells depleted of more than 50% of their intracellular free cystine by incubation in cystine-free medium reaccumulate cystine rapidly after replacement of cystine-containing medium. When cystinotic cells were depleted of their free cystine by treatment with dithiothreitol in a cystine-free medium and were then replaced in complete medium the intracellular free cystine reaccumulated rapidly, regaining its initial value in about 4 hr. The intracellular free cystine content found after 10 hr in complete medium reflected the cystine content of the medium. Cells in medium containing 0.266 mM cystine reaccumulated almost 60% more cystine than cells in medium with 0.066 mM cystine. When cystinotic and normal control fibroblasts were incubated for 2, 4, and 24 hr in either [35S]methionine or [35S]cystathionine, no radioactivity was recovered as cystine, cysteine, or glutathionine.

Cystine exodus from the granular fractions of fibroblasts, lymphoblasts, and rat liver is stimulated by ATP. We observe a similar ATP effect in leukocytes. N-ethyl maleimide (NEM), an inhibitor of the lysosomal H+-translocating ATPase (H+ATPase), eliminated the stimulation by ATP, but had no effect on basal exodus. In a series of three experiments on different days the % cystine loss in 30 min. at 37° was 54.5±1.9 (mean±SD) in pH 7.0 Hepes/sucrose buffer, 51.6±3.7 in buffer with 1 mM NEM, 76.6±4.8 in buffer with 2 mM MgATP, and 57.3±8.1 in buffer with NEM and MgATP. The initial concentration of cystine in these experiments ranged from 19 to 52 nmol cystine/unit β-hexosaminidase (cys/hex). The ATP effect disappeared when the load was increased to 1200 nmol cys/hex. The cystine loss for the same four conditions as above was 25.2, 29.8, 23.6, and 20.1%, respectively. At initial loads from 22-117 cys/hex, ATP stimulated exodus 2.2±0.6 (mean±SD, n=5) fold, whereas at loads from 800-1300 cys/hex, ATP stimulated exodus 1.1±0.2 (n=3) fold. The increased rate in the presence of ATP may represent improved binding of the cystine to the partially saturated inner transporter resulting from conformational or charge optimization brought about by the H+ATPase. The effect disappears either in the presence of the H+ATPase inhibitor NEM or when the transporter becomes saturated by high load levels.

The variations in blood ketone bodies (BKB), blood glucose (BG) and insulin were studied in 19 normal and 14 hypoglycemic children aged 4 months to 13 years during a 24 hour fast. Except in 4 patients (2 with hyperinsulinism, 2 with defect in ketogenesis), a significant and parallel increase in BKB was observed in controls and patients from Oh to 20h (in mmol/1, mean±SD) 0.20±0.16 at Oh, 1.54±1.26 at 15h, 3.05±1.45 at 20h, 3.87±1.48 at 24h. A significant progressive decrease in glucose levels was observed from Oh to 20h but not from 20h on (in mg/dl) 116±16.4 at Oh, 74.5±16 at 15h, 60±15.5 at 20h, 59.57±13 at 24h. A highly negative correlation between BKB and BG was found at each time of fasting (r≥-0.54, p -0.43, p

The recent commentary by Brown et al. contains a number of important warnings about possible dangers in the treatment of Reye's syndrome. There is, however, one additional warning which must be added. The authors speak of Reye's snydrome as a disease rather than as a syndrome. This may seem to be a minor point, but it has significant implications. A disease is an entity with a specific cause, natural history, etc. A syndrome is a group of symptoms and signs with diverse causes, natural histories, etc.

In cystinosis there is a defect in cystine efflux from the lysosomes which is expressed in both cultured fibroblasts and E.B. virus transformed lymphoblasts. Cystine efflux from lysosomes isolated in Hepes/Sucrose from control lymphoblasts is stimulated by the addition of MgATP, while efflux from lysosomes isolated from control fibroblasts is not. To clarify the role of ATP and the lysosomal ATPase in cystine efflux, we determined the pH of lysosomes isolated in simultaneous experiments from both cell types by measuring the fluorescence of intralysosomal FITC-dextran. The pH of lymphoblast lysosomes is 6.5±.05. This is lowered to 6.2±.03 by the addition of MgATP to the incubation buffer. Fibroblast lysosomes have a pH of 5.5±.07 which is not altered by the addition of MgATP. However, if the pH of fibroblast lysosomes is raised by preincubation with KCl, ATP causes acidification to near-initial values. In both cell types, there is no significant difference between cystinotic and control lysosomes in initial pH, ability to use ATP to produce an acidic pH, or the amount of proton-translocating ATPase activity. Our interpretation of these data is that cystine efflux from lysosomes requires a highly acidic intralysosomal pH. Since fibroblast lysosomes isolated in Hepes/Sucrose have a pH of 5.5 independent of ATP addition, cystine efflux is not stimulated by ATP in this system. In contrast, lymphoblast lysosomes are acidified in the presence of ATP, explaining the requirement of cystine efflux for ATP.

Ascorbic acid (0.57 mM) caused a slow decline in the free (acid-soluble) cystine content of skin fibroblasts from 6 cystinotic patients of 58.6% (range 48-75%) over a 3 day period. The cystine content then remained at this level as long as ascorbic acid was added every 24 hours. When ascorbic acid was removed, the cystine content returned to its original value in 2-3 days. Dehydroascorbic acid, the oxidation product of ascorbic acid, caused a similar reduction in cystine content of cystinotic fibroblasts. Thus, the effect of ascorbic acid is not related to its reducing properties. Cystinotic fibroblasts preloaded with 35S-cystine rapidly released 35S when treated with dithiothreitol (DTT) but not when treated with ascorbic acid. When cystinotic fibroblasts were depleted of cystine with DTT, the cystine reaccumulated 20% more slowly with dehydroascorbic acid (0.57 mM) in the media. Thus, ascorbic acid and dehydroascorbic acid appear to act by slowing the entry of cystine into the cystine pool of cystinotic fibroblasts and not by increasing the release of cystine from this pool.