Studies of Glycine Metabolism and Transport in Fibroblasts from Patients with Nonketotic Hyperglycinemia

Abstract

SummaryGlycine transport in both normal and nonketotic hyperglycinemia fibroblasts was shown to occur by a sodium-dependent system. No significant difference could be detected in either the Km's (1.4 to 2.0 mM) or the Vm,' s (6.2 to 16 nmole per mg protein per min) of the three control and three patient cell lines. Valine was a weak competitive inhibitor of glycine uptake. Ki's from both groups fell into the 5.6 to 5.8 mM range. Plasma levels of valine of one patient reached a maximum of 0.6 mM following a valine load. Glycine cleavage activity could not be detected in either control or nonketotichypergl~cinemia fibroblast lines. Serine utilization was the same in both nonketotic hyperglycinemia and control lines. SpeculationThe fibroblast lines of nonketotic hyperglycinemia patients used in our study indicate that a glycine transport defect is not the cause of the elevated cerebrospinal fluid glycine levels observed in nonketotic hyperglycinemia. The clinical valine effect is unlikely to be related to the inhibition of glycine transport by valine.Nonketotic hyperglycinemia (NKH) is a metabolic disease characterized by the early onset of hypotonia, lethargy, and myoclonic convulsions. No treatment yet devised has avoided the profound mental retardation in surviving patients. A disproportionate elevation of glycine is seen in the cerebrospinal fluid and a decrease in the activity of the glycine cleavage enzyme in brain and liver (10. 12) has been reported. However. this abnormality has also been found in the biochemically distinct disorder, ketotic hyperglycinemia (2, 12) which raises the possibility that it may not be the primary genetic defect in NKH.In some patients with NKH, valine has been shown to induce coma and markedly exacerbate the hypotonia (7. 8). This striking effect has led to the suggestion that NKH represents a defect in valine metabolism (8).More recently. Revsin and Morrow ( I 1 ) found V, . , differences in fibroblast glycine transport between NKH and control cell lines. An inhibition of glycine transport by valine was briefly noted by these authors.In our study, we investigated the influence of valine on glycine transport as a possible cause of the clinical valine effect. Glycine oxidation and serine utilization in fibroblasts were also examined. MATERIALS AND METHODSHuman diploid fibroblasts (24) from three normal and three NKH patients were grown in Eagle's minimal essential medium (13) containing 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 pg/ml).For transport studies, cells were grown on washed sterile coverslips ( 1 l x 22 mm) (14). With the fibroblasts at confluency, the 93 coverslips were rinsed twice with phosphate-buffered saline glucose (PBSG) [I30 mM NaCI, 5 mM KCI, 1.2 mM MgS04, 1 mM CaC12, 5 mM glucose. and 10 mM Na2HP04 (pH 7.4)]. In a final wash, the coverslips were left for one hr in PBSG at 37OC to minimize endogenous glycine levels.The incubation procedure was similar to that of Foster and Pardee (3). Four yl of [2-L4C]glycine (15) were added per ml of...

Normal glycine transport in cultured diploid fibroblasts from hyperglycinaemic subjects

“…Our findings agree welt with recent publications by Kelly and colleagues (1979) and Halton and Krieger (1980), who both found normal glycine transport in cells from patients with NKH, using the same cover slit method as Revsin and Morrow (1976).…”

“…These authors, however, used other competitors and this might explain the different conclusions. A graver discrepancy, however, exists when measuring transport capacity in fibroblasts from patients with NKH, where Revsin and Morrow (1976) found diminished capacity while Kelly and colleagues (1979), Halton and Krieger (1980), and we have found normal transport capacity.…”