Impairment of sympathetic activation during static exercise in patients with muscle phosphorylase deficiency (McArdle''s disease).

Static exercise in normal humans causes reflex increases in muscle sympathetic nerve activity (MSNA) that are closely coupled to the contraction-induced decrease in muscle cell pH, an index of glycogen degradation and glycolytic flux. To determine if sympathetic activation is attenuated when muscle glycogenolysis is blocked due to myophosphorylase deficiency (McArdle's disease), an inborn enzymatic defect localized to skeletal muscle, we now have performed microelectrode recordings of MSNA in four patients with McArdle's disease during static handgrip contraction. A level of static handgrip that more than doubled MSNA in normal humans had no effect on MSNA and caused an attenuated rise in blood pressure in the patients with myophosphorylase deficiency. In contrast, two nonexercise sympathetic stimuli, Valsalva's maneuver and cold pressor stimulation, evoked comparably large increases in MSNA in patients and normals. The principal new conclusion is that defective glycogen degradation in human skeletal muscle is associated with a specific reflex impairment in sympathetic activation during static exercise.     

Metabolic compensation for profound erythrocyte adenylate kinase deficiency. A hereditary enzyme defect without hemolytic anemia.

A child with hemolytic anemia was found to have severe erythrocyte adenylate kinase (AK) deficiency, but an equally enzyme-deficient sibling had no evidence of hemolysis. No residual enzyme activity was found in erythrocytes by spectrophotometric methods that could easily have detected 0.1% of normal activity. However, concentrated hemolysates were shown to have the capacity to generate small amounts of ATP and AMP from ADP after prolonged incubation. Hemolysates could also catalyze the transfer of labeled gamma-phosphate from ATP to ADP. Intact erythrocytes were able to transfer phosphate from the gamma-position of ATP to the beta-position, albeit at a rate substantially slower than normal. They could also incorporate 14C-labeled adenine into ADP and ATP. Thus, a small amount of residual AK-like activity representing about 1/2,000 of the activity normally present could be documented in the deficient erythrocytes. The residual activity was not inhibited by N-ethylmaleimide, which completely abolishes the activity of the normal AK1 isozyme of erythrocytes. The minute amount of residual activity in erythrocytes could represent a small amount of the AK2 isozyme, which has not been thought to be present in erythrocytes, or the activity of erythrocyte guanylate kinase with AMP substituting as substrate for GMP. Peripheral blood leukocytes, cultured skin fibroblasts, and transformed lymphoblasts from the deficient subject manifested about 17, 24, and 74%, respectively, of the activity of the concurrent controls. This residual activity is consistent with the existence of genetically independent AK isozyme, AK2, which is known to exist in these tissues. The cause of hemolysis in the proband was not identified. Possibilities include an unrelated enzyme deficiency or other erythrocyte enzyme defect and intraction of another unidentified defect with AK deficiency.     

Mechanisms of the acquired erythrocyte enzyme deficiencies in blood diseases.

Acquired enzymatic activity defects of erythrocyte pyruvate kinase, glucose phosphate isomerase and phosphofructokinase have been studied in patients with acute myeloid leukemias, sideroblastic refractory anemias and unclassified acquired dyserythropoiesis. 6 patients with acute myeloid leukemia had a lowered erythrocyte pyruvate kinase activity; in 5 of them the concentration of the "pyruvate kinase"-antigen was parallely decreased, in such a manner that the ratio enzyme activity/immunologic reactivity (i.e. the molecular specific activity) was normal. In 1 patient with acute leukemia, 4 with refractory anemia and 1 with acquired dyserythropoiesis the defect of the pyruvate kinase activity was associated with a normal antigen concentration (and, therefore, the molecular specific activity in whole hemolysate was lowered). The enzyme activity was restored by incubation with SH reagents in two cases and by partial purification as often as it was performed. The electrofocusing pattern of erythrocyte pyruvate kinase was normal in both these types of defects. In two patients with so-called "acquired dyserythropoiesis" an erythrocyte glucose phosphate isomerase deficiency has been detected; in both the cases it was associated with a parallel decrease of the antigen concentration. The residual enzyme had a normal electrofocusing and electrophoretic pattern and a normal heat stability; the enzyme activity could not be restored by any treatment. In 1 patient with erythroleukemia and in 1 other with acquired dyserythropoiesis the erythrocyte phosphofructokinase activity was lowered. The enzyme activity was not restored by cross incubation in isologous plasma or by the SH reagents. In one case immunologic study could be performed, indicating that the enzyme defect was mainly due to the decreased ratio of the muscle type subunit of the erythrocyte phosphofructokinase. The electrofocusing pattern of deficient phosphofructokinases was normal. Finally, we point out the probable existence of several direct mechanisms, genetic and post translational, accounting for the acquired enzyme defects of red blood cells in various blood disorders.     

Galactose-1-phosphate uridyltransferase activities in erythrocytes from a patient with galactosemia: Discrepancy between two methods

When measuring with the spectrophotometric UDP-Glu consumption test, the galactose-1-phosphate uridyltransferase (Gal-PUT) activity in erythrocyte lysates from a 22-month-old infant with a late onset form of galactosemia was found to be approximately 25% of normal. With a radiochemical assay only a very low residual activity could be detected (± 1% of normal). Preincubation of the patient's lysate with purified NADase caused a marked decrease of residual Gal-PUT activity as judged from the data obtained with the consumption test. The radiochemical assay was not influenced by a similar pre-treatment. The high level of residual activity found with the consumption test in this patient was attributed to the consumption of UDP-Glu by other reactions than Gal-PUT. Because it is a direct, simple and generally applicable assay, the radiochemical procedure is suggested to be the best method for the more detailed enzymological characterisation of the Gal-PUT deficient state in galactosemics.     

Purine Nucleoside Phosphorylase Deficiency: EVIDENCE FOR MOLECULAR HETEROGENEITY IN TWO FAMILIES WITH ENZYME-DEFICIENT MEMBERS

Purine-nucleoside phosphorylase (NP) deficiency is associated with severely defective thymus-derived (T)-cell and normally functioning bone marrow-derived (B)-cell immunity. In this study, two unrelated families with a total of three NP deficient members were investigated.High pressure liquid chromatography of the plasma of the three patients showed inosine levels greater than 66 muM. This nucleoside was absent from the plasma of their parents and control samples.NP was purified from normal human erythrocytes by affinity chromatography and an antiserum prepared in rabbits was used to study the NP variants in the two families.In family M the patient had no detectable erythrocyte NP activity and no detectable immunological-reacting material (irm) to the NP antibody. The parents, who are second cousins, had less than one-half of normal enzyme activity and approximately 14% irm attributable to a variant protein. Their electrophoretic patterns revealed a series of isozymes with slower than normal migration.In family B the patients had 0.5% residual enzyme activity and about one-half normal irm. Their electrophoretic pattern showed faintly staining bands which migrated faster than normal NP. The mother of the patients had one-half normal enzyme activity, 11% irm attributable to her variant protein, and a normal electrophoretic pattern. The father had less than one-half normal enzyme activity, equal amounts of normal and variant irm, and an electrophoretic pattern that showed increased activity of the more rapidly migrating isozyme bands.The combined use of immunological and electrophoretic techniques has shown the presence of three separate mutations; one in family M and two in family B associated with severely defective T-cell function.     

Biotinidase deficiency: the enzymatic defect in late-onset multiple carboxylase deficiency

Late-onset multiple carboxylase deficiency is characterized clinically by skin rash, alopecia, seizures and ataxia and occasionally by candidiasis and developmental delay. Biochemically, these individuals exhibit findings consistent with a combined deficiency of the biotin-dependent carboxylases. We have found that the activity of the enzyme biotinidase is also deficient in the sera of five affected children (0 to 3% of mean control activity, 5.80 +/- 0.89 nmol X min-1 X ml-1 serum), and believe that it represents the primary biochemical defect in this disease. Biotinidase catalyzes the removal of biotin from the epsilon-amino group of lysine, through which biotin is covalently bound to the four known human carboxylases, thereby regenerating biotin for reutilization. The deficient activity in our patients was not due to an inhibitor, particularly biotin. It is also not a consequence of feedback control in affected individuals under treatment with pharmacologic doses of biotin. The biotinidase activities of the parents of those children who were available for study were intermediate between deficient and normal values (46% to 65% of mean normal activity). Children lacking biotinidase activity are unable to recycle biotin, and are thus entirely dependent upon exogenous biotin to prevent deficiency. Our findings indicate that the primary biochemical defect in late-onset multiple carboxylase deficiency is in biotinidase activity which is inherited as an autosomal recessive trait.     

Intestinal β-galactosidases: II. Biochemical alteration in human lactase deficiency

Despite the high prevalence of intestinal lactase deficiency in some racial groups and in patients with intestinal disease, the biochemical defect has not been characterized.In the preceding paper normal intestine was found to have two lactases with distinctly different pH optima. Therefore, pH activity curves of homogenates from lactase-deficient intestine were studied, and the pH optimum was found to be shifted from the normal of 5.8 to 4.8. Density gradient ultracentrifugation of intestinal material from five lactase-deficient patients demonstrated absence of a lactase with pH optimum 6.0 and molecular weight 280,000. A second lactase with pH optimum 4.5 and molecular weights of 156,000 and 660,000 remained at normal levels accounting for the shift in the pH optimum in whole intestinal homogenates. In addition, three of the five patients had absence of a smaller beta-galactosidase (molecular weight 80,000) that had specificity only for synthetic substrates. Although not a lactase, this enzyme had a pH optimum identical with the missing lactase, and its activity was inhibited by lactose in a partially competitive manner suggesting that it is capable of binding lactose. It is possible that this enzyme is a precursor or fragment of the missing lactase.The residual lactase activity provided by the lactase with low pH optimum represents 20-70% of the activity of the missing enzyme, and yet these patients are not able to digest dietary lactose. Thus it appears that the residual enzyme plays no significant role in the hydrolysis of ingested lactose.     

Low therapeutic threshold for hepatocyte replacement in murine phenylketonuria.

Phenylalanine homeostasis in mammals is primarily controlled by liver phenylalanine hydroxylase (PAH) activity. Inherited PAH deficiency (phenylketonuria or PKU) leads to hyperphenylalaninemia in both mice and humans. A low level of residual liver PAH activity ensures near-normal dietary protein tolerance with normal serum phenylalanine level, but the precise threshold for normal phenylalanine clearance is unknown. We employed hepatocyte transplantation under selective growth conditions to investigate the minimal number of PAH-expressing hepatocytes necessary to prevent hyperphenylalaninemia in mice. Serum phenylalanine levels remained normal in mice exhibiting nearly complete liver repopulation with PAH-deficient hepatocytes (<5% residual wild-type liver PAH activity). Conversely, transplantation of PAH-positive hepatocytes into PAH-deficient Pah(enu2) mice, a model of human PKU, yielded a significant decrease in serum phenylalanine (<700 muM) when liver repopulation exceeded approximately 5%. These data suggest that restoration of phenylalanine homeostasis requires PAH activity in only a minority of hepatocytes.     

Determination of lysosomal enzymes in saliva. Confirmation of the diagnosis of metachromatic leukodystrophy and fucosidosis by enzyme analysis.

This paper deals with the measurement of acid hydrolases in uncentrifuged and centrifuged saliva of normal controls and the determination of some of their physical characteristics such as pH optimum, thermal stability, and residual activity after freezing and storage. From this study it appears that beta-D-glucosidase sediments totally, whereas the other enzymes show varying residual activity in the supernatant after centrifugation. In the saliva from 2 patients with metachromatic leukodystrophy and 2 patients with fucosidosis a deficiency of arylsulphatase A and alpha-L-fucosidase, respectively, was found.     

A sensitive method to assay blood complement C1- inhibitor activity

Hereditary angioneurotic edema results from deficiency of complement protein C1- inhibitor. Using a new spectrophotometric assay for C1-s esterase activity on the N-alpha-benzoyl-L-arginine ethyl ester, we describe a routinely available method for quantifying low C1- Inhibitor functional activities in EDTA-treated plasma of hereditary angioneurotic edema patients. C1- Inhibitor activity is deduced from the residual esterase activity of C1-s incubated with 20-80 microliters plasma samples. Arbitrary units (volume of sample inhibiting 50% of C1-s activity) were used to express C1- Inhibitor normal activity which was estimated as 22,500 +/- 5,000 (SD) U/l in 45 healthy individuals. The correlation with C1- Inhibitor antigen in these healthy individuals and 89 patients with varying concentrations of C1 Inhibitor ranging from 0.05-1.05 g/l was r = 0.91. Levels down to 2,000 U/l could be estimated. Specific inhibitory activity is an absolute requirement to distinguish between type I and type II hereditary angioneurotic edema.     

Erythrocyte Adenosine Deaminase Deficiency without Immunodeficiency: EVIDENCE FOR AN UNSTABLE MUTANT ENZYME

Inherited deficiency of the purine salvage enzyme adenosine deaminase (ADA) gives rise to a syndrome of severe combined immunodeficiency (SCID). We have studied a 2.5-yr-old immunologically normal child who had been found to lack ADA in his erythrocytes during New York State screening of normal newborns. His erythrocytes were not detectably less deficient in ADA than erythrocytes of ADA(-)-SCID patients. In contrast, his lymphocytes and cultured long-term lymphoid cells contained appreciably greater ADA activity than those from patients with ADA(-)-SCID. This residual ADA activity had a normal molecular weight and K(m) but was markedly unstable at 56 degrees C. His residual erythrocytes-ADA activity also appeared to have diminished stability in vivo. ADA activity in lymphoid line cells of a previously reported erythrocyte-ADA-deficient!Kung tribesman was found to contain 50% of normal activity and to exhibit diminished stability at 56 degrees C. ATP content of erythrocytes from both partially ADA-deficient individuals was detectably greater than normal (12.3 and 6.1 vs. normal of 2.6 nmol/ml packed erythrocytes). However, the dATP content was insignificant compared to that found in erythrocytes of ADA(-)-SCID patients (400-1,000 nmol/ml packed erythrocytes). The New York patient, in contrast to normals, excreted detectable amounts of deoxyadenosine, but this was <2% of deoxyadenosine excreted by ADA(-)-SCID patients. Thus, the residual enzyme in cells other than erythrocytes appears to be sufficient to almost totally prevent accumulation of toxic metabolites.     

Acute renal failure in McArdle's disease.

A case of reversible acute renal failure in an adult with myophosphorylase deficiency (McArdle's disease) is described. Myoglobinuria, possibly abetted by intravenous urography, was incriminated as the cause of impairment of renal function.A muscle biopsy specimen contained necrotic fibers and no histochemically demonstrable phosphorylase activity. Ultrastructurally, nonspecific degenerative changes were observed.     

Homocystinuria due to cystathionine synthase deficiency: the effect of pyridoxine

We investigated the effect of pyridoxine administration in three patients with homocystinuria due to cystathionine synthase deficiency. The drug decreased the plasma concentration and urinary excretion of methionine and homocystine and the urinary excretion of homolanthionine and the homocysteine-cysteine mixed disulfide. Urinary cystine rose somewhat. Oral methionine tolerance tests before and during the patients' response to pyridoxine indicated that during response they remained deficient in their capacity to convert the sulfur of methionine to inorganic sulfate, although this capacity increased somewhat. During pyridoxine response only, the methionine loads caused increased homocystinuria. There was no indication that pyridoxine stimulated an alternate pathway of metabolism. The values for specific activity of cystathionine synthase in liver biopsy specimens from two patients in pyridoxine response were 3 and 4% of the mean control value. When these patients were not receiving pyridoxine, comparable values were 2 and 1%, respectively. The hepatic enzyme activity of the mutant patients was similar to normal enzyme activity with respect to trypsin activation, heat inactivation, and stabilization by pyridoxal phosphate. Approximate estimates were made of the relation between total body capacity to metabolize methionine and hepatic cystathionine synthase activity. These estimates suggested that because of the large normal reserve capacity of cystathionine synthase, a few per cent residual activity is sufficient to metabolize the normal dietary load of methionine. Thus, small increases in residual capacity may be of major physiological importance. However, many liver biopsies would be required to establish unequivocally that such changes were due to the administration of a particular therapeutic agent rather than to biological variation. All the data in the present study are consistent with the interpretation that pyridoxine does act by causing an increase in residual cystathionine synthase activity.     

Lactosyl ceramidosis: Deficient activity of neutral β-Galactsidase in liver and cultivated fibroblasts?

Neutral beta-galactosidase was partially purified from liver of normal controls, a patient with Niemann-Pick disease type A and the previously described patient with lactosyl ceramidosis using Concanavalin A-Sepharose adsorption and Sephadex G-100 gel filtration. The partially purified fractions were essentially free of galactosyl ceramide beta-galactosidase and GM1 beta-galactosidase activities. The normal and Niemann-Pick fractions were found to hydrolyze lactosyl ceramide, in the presence of sodium taurodeoxycholate, at a pH optimum of 5.6 as well as aryl beta-galactosides and aryl beta-glucosides at pH 6.2. The corresponding fraction from the lactosyl ceramidosis liver contained only 1--4% of the normal activity towards artificial substrates and lactosyl ceramide. Cross-reacting material identical to the normal was demonstrated in this fraction with antiserum raised against purified neutral beta-galactosidase, but no activity was observed in the precipitin line when stained with naphthol AS-LC-beta-galactoside or naphthol AS-LC-beta-glucoside. A similar deficiency of neutral beta-galactosidase activity was demonstrated in cultivated fibroblasts of the patient with lactosyl ceramidosis. Following adsorption on Concanavalin A-Sepharose and anti-GM1 beta-galactosidase antibody-Sepharose conjugates and chromatography on DEAE cellulose, fibroblast lysates from the patient exhibited 3% of normal activity towards 4-methyl-umbelliferyl beta-glucoside at pH 6.2 and 12% of normal activity towards lactosyl ceramide at pH 5.6. These data suggest that neutral beta-galactosidase may have an in vivo role in the cleavage of lactosyl ceramide and that a deficiency of this activity may be related to the lactosyl ceramide accumulation observed in the patient with lactosyl ceramidosis.     

Glucose-6-phosphatase activity in liver and blood platelets of two patients with glycogen storage disease type I.

Glucose-6-phosphatase (G-6-Pase) activity in liver and blood platelets of two patients with glycogen storage disease (GSD) type I is described. Both patients had a reduced activity of G-6-Pase in liver. The km value for glucose 6-phosphate (G-6-P) of residual activity in liver of both patients was similar to that of control liver. We could not demonstrate any reduced activity of platelet G-6-Pase in the patients. Platelet G-6-Pase with our assay method seems to represent a nonspecific phosphatase activity. Our observation suggests that it is necessary to examine platelet G-6-Pase of many other patients with GSD type I to confirm that G-6-Pase deficiency can be diagnosed by enzyme assay performed on blood platelets.     

beta-Galactosidase in mucopolysaccharidoses and mucolipidoses. Deficiency of GM1 beta-galactosidase in liver and leukocytes.

beta-Galactosidase activities were studied in livers and leukocytes of mucopolysaccharidoses and mucolipidoses (I-cell disease and adult "beta-galactosidase deficiency" with macular cherry-red spots). Marked deficiency of hepatic 4-methylumbelliferyl (4MU) and GM1 beta-galactosidases was demonstrated in these diseases. Leukocyte GM1 beta-galactosidase was also deficient in mucolipidoses. The parents of the patients with I-cell disease and "beta-galactosidase deficiency" had normal beta-galactosidase activity in plasma and leukocytes, compared to the low enzyme activity in heterozygous carriers of GM1-gangliosidosis. The cause of this enzyme deficiency in these diseases is not clear at present. It seems to be affected seondarily by exgenous factors such as unknown stored materials in the cells. Mucopolysaccharides were not increased in the livers of two cases of I-cell disease and a case of "beta-galactosidase deficiency".     

The brush border membrane in hereditary sucrase—isomaltase deficiency: abnormal protein pattern and presence of immunoreactive enzyme

In a child with hereditary sucrase-isomaltase deficiency immunoreactive enzyme was present in the intact duodenal mucosa. Polyacrylamide gel electrophoresis carried out with membrane fragments of an intestinal biopsy showed an abnormal protein band without enzyme activity. The mucosa had a relatively high residual isomaltase activity which was recovered from the gel in a position suggesting higher than normal molecular weight. The results indicated that in this patient the primary structural defect was in the sucrase moiety which was enzymatically inactive. The isomaltase subunits may have aggregated into a large molecular weight complex because of unavailability of their partners. The observation also provided evidence for separate biosynthesis of the two moieties of the sucrase-isomaltase complex.     

Abnormal erythrocyte membrane protein pattern in severe megaloblastic anemia.

The erythrocyte membrane protein pattern of patients with megaloblastic anemia was determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In severe megaloblastic anemia, secondary either to folic acid or vitamin B12 deficiency, the erythrocyte membrane protein pattern was grossly abnormal, lacking bands 1, 2 (spectrin), and 3 and having several diffuse, faster migrating bands. After adequate vitamin replacement therapy, the erythrocyte membrane protein pattern returned to normal. In mild megaloblastic anemia, secondary either to folic acid of vitamin B12 deficiency, and in severe iron deficiency anemia, the erythrocyte membrane protein pattern was normal. Erythrocyte membrane protein pattern of normal membranes did not change after mixing with abnormal membranes before polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Protease activity extracted from membranes of megalocytes was not different from normal. These findings indicate that the erythrocyte membrane protein pattern is abnormal in severe megaloblastic anemia and that this abnormality is not secondary to increased activity of the endogenous erythrocyte membrane proteinase.     

Ganglioside loading of cultured fibroblasts: a provocative method for the diagnosis of the GM2 gangliosidoses

Confirmation of deficient beta-hexosaminidase activity in suspected cases of GM2 gangliosidosis may be difficult with available assay systems if the residual activity is high (as in many juvenile cases and genetic compounds). Hexosaminidase activity is normal in the AB-variant (activator protein deficiency), although GM2-ganglioside (GM2) catabolism is severely impaired. We therefore examined ganglioside degradation in intact fibroblasts in culture. Intracellular ganglioside levels were determined in cultured human skin fibroblasts grown in standard tissue culture medium or medium supplemented with mixed bovine brain gangliosides. Cellular uptake and catabolism of the added gangliosides were manifested by modest increases in the intracellular concentrations of gangliosides GT + GD and GM1 in all cells tested, and marked accumulation of GM2 in fibroblasts from patients with GM2 gangliosidoses. Intracellular GM2 increased five- to fifteen-fold in all of the GM2 gangliosidosis cell lines tested, including those from patients with infantile Tay-Sachs disease (TSD), Sandhoff disease, late infantile and juvenile variants of TSD with high residual enzyme activity, adult onset GM2 gangliosidosis, and the AB-variant. Significant GM2 accumulation did not occur in fibroblasts from patients with GM2 gangliosidosis grown in standard medium, or in normal fibroblasts grown in ganglioside enriched medium. Our method of ganglioside feeding employs commercially available materials and no special equipment. It should be useful for the confirmation of impaired GM2 catabolism in a variety of settings.