Cystinosis. Intracellular cystine depletion by aminothiols in vitro and in vivo. J G Thoene, … , D L Olson, J A Schneider J Clin Invest. 1976;58(1):180-189. https://doi.org/10.1172/JCI108448. Research Article 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. Find the latest version: https://jci.me/108448/pdf Cystinosis INTRACELLULAR CYSTINE DEPLETION BY AMINOTHIOLS IN VITRO AND IN VIVO JESs G. THOENE, ROBERT G. OSHIMA, JOHN C. CRAwHALL, DAvD L. OLSON, and JERRY A. SGHNEDER From the Department of Pediatrics, University of California, San Diego, La Jolla, California 92093 and, the Division of Nephrology, University of Southern California, School of Medicine, Los Angeles, California 90033 A B S T R A C T Certain aminothiols rapidly deplete cul- mission, free (nonprotein) cystine accumulation within tured cystinotic skin fibroblasts of their abnormally lysosomes, renal tubular dysfunction, and progressive high free (nonprotein) cystine pool. The free cystine glomerular insufficiency leading to end-stage renal failure content of these cells is reduced by over 90% in 1 h with in the first decade of life. In spite of extensive investi- 0.1 mM cysteamine. This is more rapid than previously gations (1, 2), the primary defect leading to cystine ac- known methods of removing free cystine from cystinotic cumulation and renal failure is unknown. fibroblasts. The disulfide, cystamine, is also able to The accumulation of free cystine in fibroblasts cul- deplete cystinotic cells of free cystine. tured from cystinotic patients has provided a convenient A patient with nephropathic cystinosis and end-stage in vitro test system for the evaluation of methods of renal disease was treated with cysteamine, both intra- lowering the intracellular nonprotein cystine content of venously and orally. Both methods of administration these cells. Effective methods include growth in a low- rapidly lowered the free cystine content of the patient's cystine medium (3), treatment with dithiothreitol peripheral leukocytes. (DTT)' (4, 5), and treatment with ascorbic acid (6). Study of the patient's urinary sulfur excretion did not None of these methods has been proved effective clini- conclusively determine the effect of this therapy on the cally, although DTT treatment has been shown to lower total body cystine pool. Her renal status remained at the leukocyte nonprotein cystine content in vivo (7). end stage after 1 mo of oral cysteamine, when an epi- This report shows the effectiveness of certain amino- sode of grand mal seizures prompted cessation of the thiols in producing a rapid and complete depletion of study. Determination of the proper place of aminothiol nonprotein cystine from cystinotic fibroblasts without therapy in this disease will depend upon further clinical producing apparent morphological signs of cytotoxicity. trial with patients whose kidney function has not deteri- The cystine released is utilized by the cell for GSH syn- orated to the point of irreversible change, accompanied thesis. A series of structural analogues of the aminothiol, by careful monitoring of plasma aminothiol levels. cysteamine (2-aminoethanethiol), have been investigated and allow some tentative conclusions on the relative ef- INTRODUCTION fectiveness of these compounds in depleting cystine from Nephropathic cystinosis is an inherited error of cystine cystinotic fibroblasts. A mechanism is proposed as to metabolism characterized by autosomal recessive trans- how these compounds produce cystine depletion without This work was presented in part at The Society for themselves accumulating. Pediatric Research, April 1975, and The American Society A patient with nephropathic cystinosis and end-stage for Clinical Investigation, May 1975. renal failure was treated with cysteamine, both intra- Dr. Thoene is the recipient of U. S. Public Health Service Research Fellowship 1-F22-AM-00321 from the Na- venously and orally. Both modes of administration pro- tional Institute of Arthritis, Metabolism and Digestive Dis- duced marked lowering of the peripheral leukocyte non- eases, and is also a Lalor Foundation Fellow. Dr. Oshima protein cystine content. Renal function remained un- is the recipient of a U. S. Public Health Service Research changed during a 2-mo treatment. Fellowship 1-F22-GM-04023 from the National Institute of General Medical Sciences. 'Abbreviations used in this paper: DTT, dithiothreitol; Received for publication 18 September 1975 and in revised GSH-NEM, N-ethylmaleimide adduct of glutathione; NEM, form 26 February 1976. N-ethylmaleimide. 180 The Journal of Clinical Investigation Volume 58 July 1976-180-189 METHODS cystine depletion effect of various compounds on cystinotic granules, portions of the granule suspension were added Cell culture and biochemistry. Skin fibroblasts from to tubes containing the test reagents and incubated at 25°C patients with nephropathic cystinosis and normal individuals for 30 min. Granules from 100 ,ul of the incubation mixture were established in culture and maintained as previously were recovered by centrifugation at 27,000 g for 20 min, described (8, 9). 7-9 X 0I cells were plated in 100-mm sonicated in the presence of 5 mM NEM in 10 mM po- Falcon Petri dishes (Falcon Plastics, Div. of BioQuest, tassium phosphate buffer, pH 7.4, and precipitated with 3%o Oxnard, Calif.) and harvested 72 h later while in log- sulfosalicylic acid. Cystine was measured with a cystine- phase growth. A modified Ham's F12 medium was used binding protein assay (8). Urinary sulfur and sulfate were for complete (cystine-containing) medium (8). Cystine-de- determined as previously described (14). Urinary and ficient medium was the same medium with cystine omitted. plasma amino acids were determined with a Durrum 500 Both were supplemented with 10%o fetal bovine serum. amino acid analyzer (Durrum Instrument Corp., Palo Alto, When used to supplement the cystine-deficient medium, the Calif.) (15). fetal bovine serum was dialyzed. Case report. A 7-yr-old white girl with nephropathic cys- Unradiolabeled cells were harvested by trypsinization (8) tinosis was the fourth affected of seven siblings in this after being washed three times in phosphate-buffered saline family. Three older sibs have died of renal failure due to (10) at 4°C. Cell number was determined with a Coulter cystinosis. Renal transplantation was refused by the parents, Counter, model ZBI (Coulter Electronics Inc., Hialeah, and a trial of cysteamine was therefore offered. Food and Fla.). Protein was determined colorimetrically (11) with Drug Administration approval and informed consent were bovine serum albumin as the standard. The intracellular obtained before the drug trial. nonprotein cystine content of fibroblasts and leukocytes was The patient was a small, pale child with a weight of measured by a cystine-binding protein assay (8). L-['S]- 17.6 kg (<3 percentile) and height of 107 cm (<3 per- Cystine (sp act 395 mCi/mmol and 23.2 Ci/mmol) was centile). Slight frontal bossing and uremic breath were obtained from Amersham/Searle Corp. (Arlington Heights, the only other positive physical findings. Her pulse was Ill.) and New England Nuclear (Boston, Mass.), respec- 85, her respiration 16, and her blood pressure 114/74. tively. Purity of labeled cystine was greater than 98% when Initial laboratory values included a blood urea nitrogen tested by high-voltage electrophoresis, followed by radio- of 145 mg/100 ml, serum creatinine of 6.5 mg/100 ml, crea- chromatogram scan (see below). Cysteamine and its ana- tinine clearance of 8.7 ml/min per 1.73 m2, calcium of 9.9 logues were the best available grade (over 97% purity) mg/100 ml, phosphorus of 11.6 mg/100 ml, white blood cell and obtained from either Calbiochem (San Diego, Calif.) count of 5,600/mm3, reticulocytes of 1.1%o, hemoglobin 6.7 or Aldrich Chemical Co., Inc. (Milwaukee, Wis.). Omni- mg/100 ml, and a hematocrit of 20. Serum electrolytes, liver fluor was purchased from New England Nuclear. functions, white blood cell differential, and platelet count In studies with labeled material, Petri dishes of fibro- were within normal limits. Long-bone X-rays demonstrated blasts in log-phase growth were washed four times in minimal changes of renal osteodystrophy. Ophthalmological phosphate-buffered saline at 4°C and harvested by direct examination showed the typical keratopathy and retinopathy freezing of the cells in the culture dish on a dry ice-ethanol of cystinosis (16). During the study period, the patient was bath in the presence of 5 mM N-ethylmaleimide (NEM) maintained on her previous regimen of oral aluminum hy- in 10 mM potassium phosphate buffer at pH 7.4. The cells droxide (Amphojel, Wyeth Laboratories, Div., American were then thawed briefly at 60°C and detached from the Home Products Corp., Philadelphia, Pa.), sodium and po- surface with a rubber scraper at 25°C. The initial cell lysate, tassium citrate solution, vitamin D, (dihydrotachysterol) plus a 2-ml H20 wash, were combined and lyophilized.