Familial hyperlysinemia with lysine-ketoglutarate reductase insufficiency Joseph Dancis, … , Rody P. Cox, Norman C. Woody J Clin Invest. 1969;48(8):1447-1452. https://doi.org/10.1172/JCI106110. Research Article Fibroblasts grown in tissue culture from the skin of normal subjects have lysine-ketoglutarate reductase activity (lysine: α- ketoglutarate: triphosphopyridine nucleotide (TPNH) oxidoreductase (ε-N-[L-glutaryl-2]-L-lysine forming)). The activity of the enzyme is considerably reduced in the skin fibroblasts grown from three siblings with hyperlysinemia. The high concentrations of lysine in the blood of these patients, the previous demonstration in the intact subject of a reduction in the ability to degrade lysine, and the present demonstration of diminished lysine-ketoglutarate reductase activity, accurately define the metabolic defect and establish the saccharopine (ε-N-[L-glutaryl-2]-L-lysine) pathway as the major degradative pathway for lysine in the human. Find the latest version: https://jci.me/106110/pdf Familial Hyperlysinemia with Lysine- Ketoglutarate Reductase Insufficiency JOSEPH DANCGS, JOEL HurziER, RODY P. Cox, and NORMAN C. WOODY From the Departments of Pediatrics and Medicine, New York University School of Medicine, New York 10016, and the Department of Pediatrics, Tulane Medical School, New Orleans, Louisiana 70112 A B S T R A C T Fibroblasts grown in tissue culture from made it possible to investigate more specifically the en- the skin of normal subjects have lysine-ketoglutarate zyme defect. In the present study, it has been demon- reductase activity (lysine: a-ketoglutarate: triphospho- strated that fibroblasts grown in tissue culture from skin pyridine nucleotide (TPNH) oxidoreductase (c-N- [L- biopsies obtained from normal subjects convert lysine to glutaryl-2] -L-lysine forming)). The activity of the en- saccharopine. In comparison, the lysine-ketoglutarate zyme is considerably reduced in the skin fibroblasts reductase activity was considerably reduced in the skin grown from three siblings with hyperlysinemia. The fibroblasts of the three siblings with the disease. The re- high concentrations of lysine in the blood of these pa- sults accurately define the enzyme defect in the disease tients, the previous demonstration in the intact subject and establish the saccharopine pathway as the major of a reduction in the ability to degrade lysine, and the degradative pathway for lysine. present demonstration of diminished lysine-ketoglutarate reductase activity, accurately define the metabolic defect METHODS and establish the saccharopine (c-N- [L-glutaryl-2] -L- Materials lysine) pathway as the major degradative pathway for lysine in the human. L-lysine-U-14C was purchased from New England Nuclear Corp., Boston, Mass. A trace radioactive contaminant with electrophoretic properties similar to saccharopine was re- INTRODUCTION moved by passage through a column of Rexyn 206 (OH-), Fisher Scientific Company, New York. The resin was pre- In 1964, a 4J yr old girl was reported with mental retar- pared by cycling with 3 N HCl and 1 N ammonium hydrox- dation, excessive lysinuria, and hyperlysinemia (1). ide and then was left in the hydroxyl phase. Under these Since then two additional siblings and a first cousin have conditions, lysine was eluted from the column with water. been discovered to have a similar metabolic abnormality. Cab-O-Sil, fumed silica, grade M5, was obtained from Cabot Corporation, Boston, Mass. Saccharopine was ex- The metabolism of lysine has been studied in vivo in two tracted from yeast by methods previously described (5). of the siblings and evidence has been presented that a defect exists in the degradative pathway for lysine prob- Tissue culture ably at the first metabolic step (2). Skin biopsies were obtained from the three patients with The recent demonstration of saccharopine (E-N-[L- hyperlysinemia and grown in monolayer in Waymouth's glutaryl-2]-L-lysine) formation from lysine in the rat medium containing 10%o calf serum and antibiotics (peni- (3, 4) and in the human (5), and the development in cillin 50 U/ml, streptomycin, 50 /Ag/ml, and kanamycin 30 this laboratory of a microassay for the responsible en- ,Ag/ml). Foreskins obtained at circumcision of newborn infants were cultured as normal controls. Subcultures of the zyme, lysine-ketoglutarate reductase, (lysine: a-ketoglu- explants were permitted to grow to confluence in 4-oz glass tarate: triphosphopyridine nucleotide (TPNH) oxido- bottles. At the time of assay, the normal cells had pro- reductase (E-N-[L-glutaryl-2]-L-lysine forming)) have liferated for 10-18 generations and the hyperlysinemics for 16-20 generations. Dr. Dancis is Career Investigator, the National Institute of Child Health and Human Development. Dr. Cox is Enzyme assay Career Scientist, Health Research Council of the City of New York. The fibroblasts from four bottles, representing a total of Received for publication 17 January 1969 and in revised approximately 4 million cells, were harvested by replacing form 3 April 1969. the nutrient medium with 5 ml of a 15: 85 v/v mixture of The Journal of Clinical Investigation Volume 48 1969 1447 300F- CONTROL G.G. PATIENT Al Orbigin Origin 2001 - A _I B V A B CONTROL M.M. PATIENT 11 z 0LS 2001 cLJ tn 100 z _m1m B m B 0 (.. L - CONTROL SS. PATIENT R.I. A - A / m B SCAN DIRECTION- FIGURE 1 Radioactive pattern after high-voltage electrophoresis of incubation media. Disrupted skin fibroblasts were incubated with lysine-14C. The incubation medium was applied to paper and subjected to high-voltage electrophoresis. Lysine migrates towards the cathode, and has been omitted from the radioactive scan. The control fibroblasts produce a radioactive peak corresponding in mobility to an internal standard of sac- charopine (stippled bar), not evident after incubation with fibroblasts from hyperlysinemic subjects. sodium phosphate, 0.11 mole/liter, pH 7.4, and 0.15 M NaCl, pH 6.7. A lower pH was used than that previously reported and removing the cells from the bottle with a rubber (5) to better separate an unidentified radioactive peak (see policeman. The suspension was centrifuged at 2000 g for 15 Fig. 1, peak A) from saccharopine. The strip was surveyed min, and the supernatant discarded. The cells were stored in a Nuclear-Chicago Actigraph III strip counter. The at -15'C for 4 days or more before testing. saccharopine peak was located by removing a 2 mm wide For assay, the cells were thawed in a 230C water bath strip from the paper and developing with ninhydrin. The and 0.15 ml of 0.05 M KC1 was added. The fibroblasts were remainder of the saccharopine area was removed, cut into then disrupted by cycling five times between a dry-ice bath small pieces with scissors, and eluted by shaking for 15 mmi and the water bath over a period of 30 min, aided by occa- in 5 ml of water at 60'C. The process was repeated and sional stirring with a glass rod. To each sample of disrupted the eluates pooled, filtered, and evaporated to dryness under cells was added the following incubation mixture: L-lysine- vacuum. The residue was dissolved in 1 ml of citrate U-14C, 0.5 juc, 1.0 umole; MgCl2, 0.05 /moles; potassium buffer, pH 2.8.1 a-ketoglutarate, 2 etmoles; potassium phosphate, pH 7.1, 10 Column chromatography. Approximately 0.2 jtmoles of /Amoles; TPNH, 1.5 Amoles; and water to a final volume methionine was added to the sample as a marker and the of 0.5 ml. Incubation was with agitation for 60 min at mixture applied to a Phoenix Micro Analyzer with a 0.9 30'C under a stream of nitrogen. X 15 cm column containing Spherix resin XX907-10-OPKU. To terminate the incubation, 5 tmoles of saccharopine in Elution was with 0.2 M sodium citrate buffer, pH 3.25,1 and 0.05 ml water was added to the incubation mixture and the a flow rate of 1 ml/min. The saccharopine peak appeared reaction tubes were placed in a boiling water bath for at 63 min. 5 min. The tubes were centrifuged while hot, and the super- Determination of radioactivity. The saccharopine peak natant removed for analysis. The amount of protein in the was collected at the outflow of the colorimeter into scintil- precipitate was measured by dissolving the precipitate in 1.0 lation counting vials in 1-min aliquots. The ninhydrin color N NaOH and assaying the protein content by the method was bleached by the addition of 0.1 ml 30%o hydrogen per- of Lowry (6). This provided an indication of the number oxide to each vial, and the contents evaporated to dryness of cells incubated. in a water bath at 600C. The residue was dissolved in 0.5 ml water. To this was added 11 ml methanol, approximately Analysis 270 mg Cab-O-Sil (to produce a gel), and 9 ml of the scintillant mixture containing (per liter) : 2,5-diphenyloxazole High voltage electrophoresis. 0.1 ml of the incubation medium was applied to Whatman 3MM paper and subjected The composition was that described by Hamilton (7) to 2000 v for 3 hr in 0.05 M potassium phosphate buffer, for buffer, pH 2.875, with the pH corrected by titration. 1448 J. Dancis, J. Hutzler, R. P. Cox, and N. C. Woody PATIENT Ii. 0.5 C)~~~~~~~~~~~~~~~ at' ~~~~~~~~~~~~~C 0~~~~~~~~~~~~~~~~~~~~0 = 2 2002 0 ~~~~~~~~~~~~~~~~~00 75 65 55 45 75 65 55 45 Minutes FIGuRE 2 Column chromatography of presumed saccharopine. After high-voltage electrophoresis (Fig. 1), the saccharopine area was eluted and analyzed by column chromatography. The ninhydrin optical density (continuous line) is indicated by the left ordinate; radioactivity by the bar graph and the right ordinate. Methionine was added as an internal standard and appears to the left of the sac- charopine peak.