Lysine-ketoglutarate reductase and saccharopine dehydrogenase activities are distributed widely in human tissues.28,29 The skin fibroblast grown in tissue culture proved a convenient approach to study of the biochemical defect. Both enzyme activities were grossly deficient in patients with familial hyperlysinemia.6,15 Retrograde splitting of saccharopine to yield lysine also was greatly reduced. It is uncertain whether this reaction is catalyzed by a distinct enzyme, saccharopine oxidoreductase, or reflects the reversibility of lysine-ketoglutarate reductase activity. Liver obtained at autopsy from a hyperlysinemic patient had the same enzyme deficiencies.6
The magnitude of the defect in lysine metabolism was estimated in two subjects by administering [14C]lysine and collecting exhaled 14CO2.30 Under conditions of normal dietary intake, the amount of labeled carbon dioxide collected from two hyperlysinemic patients was about 10 percent of that in two control subjects (Fig. 86-2). Furthermore, the controls had a reserve capacity to handle excesses of lysine. Following administration of an acute load of lysine, the amount of lysine degraded to CO2 increased twofold without increasing the fraction excreted into the urine.
Cumulative excretion of 14CO2 following administration of [U-14C]lysine. [14C]Lysine was injected into two control and two hyperlysinemic subjects, and the 14CO2 expired was measured. The control subjects also were loaded with 150 mg/kg of the lysine base prior to injection of [14C]lysine and the measurements repeated. The cumulative excretion of radioactive CO2 is presented as a percentage of administered radioactivity. (From Woody et al.,30 by permission of American Journal of Diseases of Children.)
An unusual patient with familial hyperlysinemia, presenting with “cystinuria,” was detected by careful examination of urine.31 Cystine overlies saccharopine in the usual chromatographic systems. By oxidizing cystine to cysteic acid, it was revealed that the patient had saccharopinuria rather than cystinuria. Excesses of saccharopine were unexpected because saccharopine is distal to the defect in lysine-ketoglutarate activity. To confirm this observation, the tentatively identified saccharopine was isolated from the urine and heated to 110°C for 5 hours. A new compound appeared with the chromatographic characteristics of pyrosaccharopine. Of an additional six subjects with the double enzymatic activity defect characteristic of familial hyperlysinemia, four had a detectable saccharopinuria. A reasonable explanation of this unexpected finding is that residual lysine-ketoglutarate reductase activity permits the synthesis of small amounts of saccharopine, and a more complete defect in saccharopine dehydrogenase activity prevents its metabolic disposal.
The initial observation is generally an impressive lysinuria detected during biochemical studies of a patient with presumed metabolic disease, often with neurologic symptoms. Measurement of plasma amino acid levels shows the lysine to be considerably elevated, regularly exceeding 680 mM in classic cases and often reaching twice that concentration. Screening of other family members may reveal asymptomatic individuals with the same biochemical findings. Both sexes are affected, and consanguinity of parents has been reported, indicating autosomal recessive inheritance. Confirmation of the diagnosis is by enzymatic studies on the skin fibroblast.
Normal skin fibroblasts effectively degrade lysine to carbon dioxide. It was therefore possible to reproduce the studies originally performed in vivo by incubating skin fibroblasts with [14C]lysine.15 In skin fibroblasts from seven subjects with enzymatically confirmed diagnoses of familial hyperlysinemia, 5 to 10 percent of the normal amount of radioactive CO2 was liberated (Table 86-1). In the presence of significant hyperlysinemia in the patient, a reduction in the degradation of lysine to CO2 can be assumed to involve a defect in either or both of the first two enzymatic steps in the saccharopine pathway. Hyperlysinemia has not been reported in defects below α-aminoadipic acid or as the result of defects in the pipecolic acid pathway.
Table 86-1: Metabolism of Skin Fibroblasts* |Favorite Table|Download (.pdf) Table 86-1: Metabolism of Skin Fibroblasts*
|Hyperlysinemia Patients ||Lysine-Ketoglutarate Reductase† ||Saccharopine Dehydrogenase‡ ||CO2 Evolution§ (Lysine-2-14C) |
|1 ||0 ||0 ||— |
|2 ||0 ||0 ||0.08 |
|3 ||27 ||0 ||0.17 |
|4 ||0 ||0 ||0.03 |
|5 ||4 ||0 ||0.27 |
|6 ||26 ||9 ||0.12 |
|7 ||35 ||5 ||0.06 |
|Mean ||13 ||2 ||0.12 |
|Range ||0–35 ||0–9 ||0.03–0.27 |
|Controls || || || |
|Number ||8 ||6 ||12 |
|Mean ||357 ||95.3 ||2.75 |
|Range ||240–402 ||63–164 ||0.92–6.0 |
Specific diagnoses require assays for lysine-ketoglutarate reductase and saccharopine dehydrogenase activities. These tests are not available in most laboratories, and the latter requires a substrate that is not commercially available. Fortunately, there should be few instances in which these specific assays are clinically necessary.
Skin fibroblasts are grown to confluence. Cells from about 38 cm2 of growing surface are transferred to a flat-bottomed vial, the medium removed following centrifuging, and replaced with 0.225 ml of Krebs-Ringer phosphate buffer. To this is added 0.025 ml of DL-[14C]lysine labeled in the 2 position, 20 mCi/ml in 0.08 M NaCl. The cells are incubated for 4 hours at 35°C under oxygen with gentle agitation. CO2 is collected in a center well containing 0.1 ml 1 N NaOH. The well contents are transferred to a scintillant to determine radioactivity.15 Prior to incubation, the radioactive substrate is freed of volatile impurities by bubbling with N2 at pH 3.
Fibroblasts are disrupted by repetitive freeze-thawing. Approximately 4 million cells are incubated with L-[U-14C]lysine (0.5 M, 1.0 mmol), MgCl2 (0.05 mmol), potassium α-ketoglutarate (2 mmol), potassium phosphate, pH 7.1 (10 mmol), NADPH (1.5 mmol), and water to a final volume of 1 ml.5,6 Incubation is for 60 minutes, with shaking, at 30°C under a stream of N2. Incubation is terminated by adding 5 mmol saccharopine in 0.05 ml of water and placing the tubes in boiling water for 5 minutes. The reaction mixture is centrifuged and the supernatant subjected to high-voltage electrophoresis. The saccharopine area is eluted, and saccharopine is isolated by ion-exchange chromatography. Radioactivity is measured, and the synthesis of saccharopine is calculated.
The reaction mixture contains 0.1 ml sonicated mitochondria from skin fibroblasts, 0.5 mmol NAD+ in 0.02 ml Tricine-NaOH buffer, pH 8.9, saccharopine [U-14C]glutaryl, 0.1 mCi, 0.1 mmol in water to 0.25 ml.5,6 Incubation is with agitation for 60 minutes at 25°C. The reaction is stopped by adding 0.1 ml of 10 mM glutamate solution and 0.05 ml of 1 N HCl. Radioactive glutamic acid is measured by reacting with glutamic decarboxylase. The substrate, radioactive saccharopine, is synthesized as described previously.
Several of the patients with familial hyperlysinemia were detected as a result of diagnostic studies for neurologic damage and mental retardation. The relation of the metabolic defect to the clinical manifestations was therefore uncertain. To avoid this bias, a study was conducted of patients identified during routine newborn screening or because of family surveys of affected individuals.32 Ten subjects were located who met these criteria and in whom the diagnosis of familial hyperlysinemia had been confirmed by enzymatic assays of skin fibroblasts. In none was any damage observed that was attributable to the hyperlysinemia.
One patient was particularly impressive in confirming the absence of toxicity of extremely high concentrations of lysine. A normal infant was born to a woman with familial hyperlysinemia. Lysine is rapidly transferred across the placenta, establishing levels in fetal blood that are slightly higher than in the mother. It can be safely assumed, therefore, that the fetus was exposed to severe hyperlysinemia during the susceptible periods of development without ill effect. Periodically, there are reports of familial hyperlysinemia associated with neurologic deficits34 or other disorders; however, these associations appear to be fortuitous.
Similar information is not available for the variant, saccharopinuria.
The preceding observations make it clear that hyperlysinemia of considerable magnitude can be tolerated without ill effect. They do not establish that hyperlysinemia is always benign. The question of dietary control therefore must be addressed.
Lysine is present in high concentration in most natural foods. A simple low-lysine diet cannot be devised. By reducing protein intake from the high levels that are customary in American diets, it is possible to lower the plasma lysine concentration from approximately 1400 mM to about 680 to 800 mM. Further reductions toward normal can be accomplished only by substituting a mixture of purified amino acids restricted in lysine for dietary protein.
The weight of the evidence at present is against subjecting the family to the financial and psychological burdens of strict dietary control. Some parents and physicians have chosen to limit protein intake; most have avoided any restrictions.
In 1968, a retarded 22-year-old woman was reported with saccharopinuria and a less severe lysinemia.33 It appeared reasonable at that time to attribute the metabolic defect to the second step in lysine degradation, saccharopine dehydrogenase, with familial hyperlysinemia resulting from a deficiency in the first step, lysine-ketoglutarate reductase. The subsequent recognition that both enzyme activities were defective in familial hyperlysinemia and that saccharopinuria also was observed made the distinction between the two entities less certain. It should be emphasized, however, that the magnitude of the saccharopinuria is considerably less in familial hyperlysinemia and that it is overshadowed by the lysinuria, whereas the reverse was true in the patient with saccharopinuria. The lysine/saccharopine ratios in urine have ranged from 56 to 185 in familial hyperlysinemia,31 whereas it was 0.33 in the reported case of saccharopinuria. Enzyme studies done on the patient's fibroblasts at a later date revealed that the lysine-ketoglutarate reductase activity was reduced to one-third the normal level, and no saccharopine dehydrogenase activity was detected.35 Given the many significant similarities between familial hyperlysinemia and saccharopinuria, it would appear more useful to consider the latter a variant rather than a discrete entity. A second case of saccharopinuria has been reported in which the enzymatic defects have not been as clearly defined.36 Recently, a third patient with prominent saccharopinuria as a variant form of familial hyperlysinemia was described in Japan.36a
The diagnosis of saccharopinuria was made in each instance in the course of investigations of neurologic deficits. The relation of the metabolic anomaly to the symptoms is therefore uncertain.
At the molecular level, the relation between familial hyperlysinemia and saccharopinuria may be explained on the basis of polarity mutations of a bifunctional enzyme. Defects near the origin of the protein are likely to affect both enzyme activities, whereas those nearer the termination may preferentially affect saccharopine dehydrogenase. Whether a similar mechanism applies to orotic aciduria types I and II is not known. Transferase and decarboxylase activities of the bifunctional UMP synthase are differentially affected by conformational changes in the protein (see Chap. 113).