Tissue levels of glycolytic enzymes in phosphoglycerate kinase deficiency

Phosphoglycerate kinase deficiency is a rare, X-linked disorder associated with a severe haemolytic anaemia. In general the deficiency has been demonstrated only in erythrocytes and leucocytes. However, in a subject with this condition, the activity of phosphoglycerate kinase in lymphocytes and platelets was also shown to be less than 5% of the normal value. Following the death of this subject in 1979, the deficiency was also found to occur in tissue samples of brain, skeletal muscle, liver and cardiac muscle, obtained at the autopsy. Values for phosphoglycerate kinase were of the order of 0.5–5% of normal controls. Other glycolytic enzymes which were tested were hexokinase, pyruvate kinase, enolase and 2-phosphoglyceromutase. In general, values for these enzymes were either normal or slightly raised.     

Phosphoglycerate kinase deficiency: biochemical studies on hair follicles

A fluorimetric procedure for the determination of phosphoglycerate kinase in single human hair follicles is described. Enzyme studies on different parts of hair follicles after dissection show that the distribution of glucose-6-phosphate dehydrogenase matches that of phosphoglycerate kinase. Glucose-6-phosphate dehydrogenase can therefore be used as a reference enzyme to compensate for differences in hair follicle sizes. It was shown that the variation in the values found in individual hair follicles is improved by relating phosphoglycerate kinase to glucose-6-phosphate dehydrogenase activity. In areas of the world where glucose-6-phosphate dehydrogenase deficiency occurs frequently, an autosomally inherited reference enzyme may be preferred. It is shown that 6-phosphogluconate dehydrogenase is useful in this respect. Upon storage a gradual drop in the activity of all three enzymes was observed, but the rate of decrease was about equal: the enzyme activity ratio was, therefore, almost unaffected for a period of one week. This allows the determination of phosphoglycerate kinase even in mailed hair follicles.     

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.     

Elevated red blood cell 2,3-diphosphoglycerate levels in black blood donors

Mean levels of 2,3-diphosphoglycerate (DPG) were significantly increased in erythrocytes (RBC) from 43 nonanemic black blood donors (4.80 +/- 0.06 micromoles/l RBC) compared with 22 white donors 4.47 +/- 0.08 micromoles/l RBCs from eight of the 12 black donors with DPG levels greater than 5 micromoles/l RBC. Although a potentially hemolytic disorder could be defined in four (AS hemoglobin, beta-Thalassemia minor, G6PD deficiency), reticulocyte counts were normal. However, when RBCs from the subgroup were compared to RBCs from an additional 25 unselected white donors, the following suggested an abnormally large population of young RBCs in the subgroup: 1) normal or elevated RBC-ATP with normal serum phosphate level; 2) significantly increased activities of RBC age-dependent enzymes hexokinase (p less than 0.02), pyruvate kinase (p less than 0.05), and glutamicoxaloacetic transaminase (p less than 0.01), with normal activity of phosphoglycerate kinase, an age-independent enzyme; 3) decreased dense (older) RBCs as determined by sedimentation in phthalate esters. Since DPG is increased in young RBCs and falls as the RBC ages, loss of older relatively DPG depleted RBCs due to shortened survival could account for the elevated DPG levels seen in the subgroup.     

Erythrocyte glutathione synthetase deficiency leads not only to glutathione but also to glutathione-S-transferase deficiency.

Glutathione synthetase (GSH-S) is one of the two known hereditary causes of glutathione deficiency. We describe a family whose two children have hemolytic anemia. The children's erythrocytes lack GSH and are severely deficient in GSH-S activity. No neurologic findings or 5-oxoprolinuria were present. A concurrent deficiency of glutathione-S-transferase (GST) was also detected in the erythrocytes. Residual glutathione could be detected in the erythrocytes using a sensitive cycling assay. The deficiency was found to be most severe in reticulocyte-depleted preparations. The GSH-S activity of the erythrocytes of the parents was one-half normal, while the glutathione S-transferase activity was normal. We conclude that the primary defect is one of GSH-S. Glutathione stabilizes GST in vitro, and it is assumed that the deficiency of GST in the erythrocytes of the patients is due to the instability of this enzyme in the absence of adequate intracellular GSH levels.     

Hereditary complete deficiency of lactate dehydrogenase H-subunit

We report the second known case of a patient, a 45-year-old Japanese woman, with hereditary complete deficiency of lactate dehydrogenase (LDH; EC 1.1.1.27) H-subunit. Total LDH activity in her serum was abnormally low (35 U/L, normal reference interval 195-360). LDH activity in her erythrocytes was also low, but all the other glycolytic enzyme activities in her erythrocytes were within normal limits. Electrophoresis of her serum, erythrocytes, lymphocytes, thrombocytes, and saliva showed only one band, the LDH M4 isoenzyme. LDH activities in her saliva and lymphocytes exceeded the reference interval. Her erythrocytes contained fructose 1,6-diphosphate 26 mumol/L (normal range 4-13), dihydroxyacetone phosphate 75 mumol/L (normal range 8-22), glyceraldehyde 3-phosphate 57 mumol/L (normal range 4-14), and pyruvate 45 mumol/L (normal range 31-63). The family study of three generations showed that this deficiency was inherited in an autosomal recessive mode.     

Deoxyguanosine Triphosphate as a Possible Toxic Metabolite in the Immunodeficiency Associated with Purine Nucleoside Phosphorylase Deficiency

Purine nucleoside phosphorylase (PNP) deficiency is associated with a severe defect in thymus-derived (T)-lymphocyte function combined with normal bone marrow-derived (B)-lymphocyte function. To investigate the role of this enzyme deficiency in the resulting immune dysfunction, we measured the levels of ribonucleoside and deoxyribonucleoside triphosphates in erythrocytes from two unrelated PNP-deficient, T-lymphocyte-deficient patients. Both PNP-deficient patients have abnormally high levels of deoxyguanosine triphosphate (deoxy-GTP) in their erythrocytes (5 and 8 nmol/ml packed erythrocytes). In contrast, normal controls and adenosine deaminase-deficient, immunodeficient patients do not have detectable amounts of deoxyGTP (<0.5 nmol/ml packed erythrocytes). We propose that deoxyguanosine, a substrate of PNP, is the potentially lymphotoxic metabolite in PNP deficiency. The mechanism of toxicity involves phosphorylation of deoxyguanosine to deoxyGTP, which acts as a potent inhibitor of mammalian ribonucleotide reductase.     

Human erythrocyte hexokinase deficiency. Characterization of a mutant enzyme with abnormal regulatory properties.

In the erythrocytes of a patient with hereditary nonspherocytic hemolytic anemia, a homozygous expression of hexokinase deficiency was detected. The mutant enzyme was characterized by normal kinetic parameters with respect to its substrates, glucose and MgATP2-, normal pH optimum, normal heat stability at 40 degrees C, but abnormal behavior with respect to its regulation by glucose-1,6-diphosphate and inorganic phosphate, and an altered electrophoretic pattern. Interpretation of the results revealed the presence of two different hexokinases type I in normal human erythrocytes: one enzyme with a high affinity for glucose-1,6-diphosphate, the inhibition of which is regulated by inorganic phosphate; and another enzyme with a lower affinity for the inhibitor, not regulated by inorganic phosphate. The former enzyme was not detectable in the erythrocytes of the patient, whereas the presence of the latter enzyme could be demonstrated.     

The first case of a complete deficiency of diphosphoglycerate mutase in human erythrocytes.

An inherited and complete deficiency of diphosphoglycerate mutase was discovered in the erythrocytes of a 42-yr-old man of French origin whose blood hemoglobin concentration was 19.0 g/dl. Upon physical examination he was normal with the exception of a ruddy cyanosis. The morphology of his erythrocytes was also normal and there was no evidence of hemolysis. The erythrocyte 2,3-diphosphoglycerate level was below 3% of normal values and, as a consequence, the affinity of the cells for oxygen was increased. Diphosphoglycerate mutase activity was undetectable in erythrocytes as was that of diphosphoglycerate phosphatase. The activities of all the other erythrocyte enzymes that were tested were normal except for nomophosphoglycerate mutase which was diminished to 50% of the normal value. The levels of reduced glutathione, ATP, fructose 1,6-diphosphate, and of triose phosphates were elevated, whereas those of glucose 6-phosphate and fructose 6-phosphate were decreased. This report sheds new light on the role of diphosphoglycerate mutase in the metabolism of erythrocytes.     

Human β-mercaptopyruvate sulfurtransferase: Distribution in cellular compartments of blood and activity in erythrocytes from patients with hematological disorders

The distribution of mercaptopyruvate sulfurtransferase (EC 2.8.1.2) in human blood cell compartments has been studied. Almost all of the enzyme activity in whole blood was confined to erythrocytes, but the activity per litre cell volume was higher in platelets than in erythrocytes. Leukocytes contained very low concentrations of the sulfurtransferase and plasma was devoid of activity. The enzyme activity of blood hemolysates from normal subjects and patients with various erythrocyte disorders was also determined. Significantly higher sulfur transferase activity was found in male subjects in comparison with females. The enzyme activity per litre erythrocytes was increased in blood from patients with iron deficiency anemia, whereas less clear-cut differences with respect to normals were found in other diseases studied.     

Lead poisoning: association with hemolytic anemia, basophilic stippling, erythrocyte pyrimidine 5'-nucleotidase deficiency, and intraerythrocytic accumulation of pyrimidines.

Lead intoxication is accompanied by an acquired deficiency of erythrocyte pryimidine-specific, 5'-nucleotidase. Genetically determined deficiency of this enzyme is associated with chronic hemolysis, marked basophilic stippling of erythrocytes on stained blood films, and unique intraerythrocytic accumulations of pyrimidine-containing nucleotides. The present report documents that lead-induced deficiency when sufficiently severe gives rise to findings similar to the hereditary disorder. Whereas pyrimidine-containing nucleotides are virutally absent in the erythrocytes of normal and reticulocyte-rich blood, 12% of erythrocyte nucloetides in the blood of a patient with lead intoxication contained cytidine. Nucleotidase activity was about 25% that in normal erythrocytes and 15% or less of that expected in comparable reticulocyte-rich blood. The distribution of nucleotidase activity in patient erythrocytes is unknown, and much more severe deficiency could have been present in subsets of the cell populations analyzed. The findings indicate that the hemolytic anemia and increased basophilic stippling characteristic of certain cases of lead intoxication may share a common etiology with essentially identical features of the genetically determined disorder.     

Complex Müllerian malformation without any present classification: Unilateral ovarian and tubal absence with an arcuate uterus

Müllerian duct anomalies are known to cause infertility and reproductive problems. The true incidence of such abnormalities is not well defined. The most widely accepted method of classification for a Müllerian duct anomaly is the American Society of Reproductive Medicine classification (1988). However, there are some rare anomalies inconsistent with the current classification. Herein, we report a rare case of Müllerian duct anomaly, unilateral ovarian and tubal absence with an arcuate uterus. The failure of the Müllerian ducts to canalize can also lead to the development of a unicornuate uterus and adnexal agenesis. An arcuate uterus indicates incomplete septal absorption after normal fusion of the Müllerian ducts. Therefore, its coexistence with adnexal absence and an arcuate uterus is considered to be extremely unlikely.     

A modification of the fluorescent stain method for identifying a deficit of erythrocyte phosphoglycerate kinase in mass screening]

The developed modification may be used in clinical practice as a diagnostic test and in mass screenings for phosphoglycerate kinase deficiency: more than a hundred tests may be made in a day. If blood samples have to be sent by mail, their quality is unchanged for up to 7 days.     

Lentiviral Gene Therapy Using Cellular Promoters Cures Type 1 Gaucher Disease in Mice

Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease.     

Adenine phosphoribosyltransferase deficiency: a previously undescribed genetic defect in man

A deficiency of adenine phosphoribosyltransferase (A-PRTase) is described in four members in three generations of one family. A-PRTase is coded by an autosome and the mutants described in this report are heterozygotes for this enzyme defect. The level of enzyme activity in these heterozygotes was inappropriately low, ranging from 21 to 37% of normal rather than the expected 50% of normal. Examination of various physical and chemical properties of the A-PRTase obtained from the mutant heterozygotes failed to reveal differences from the normal enzyme. These patients have no discernable abnormality in uric acid production despite the finding that patients with a deficiency of a closely related enzyme, hypoxanthine-guanine phosphoribosyltransferase, invariably produce excessive quantities of uric acid. A relationship of the A-PRTase deficiency to the disturbance in lipoprotein metabolism observed in the propositus has not been firmly established. Possible manifestations of the homozygous form of this enzyme deficiency will require identification of such individuals in the future.     

A combined system for the study of glutathione metabolism in erythrocytes.

A combined system for measuring glutathione stability, hexose monophosphate shunt activity, glycine incorporation into glutathione, glucose consumption and lactate formation in erythrocytes has been devised. In severe glucose-6-phosphate dehydrogenase deficiency associated with chronic hemolytic anemia, extremely small amounts of glucose were metabolized via the hexose monophosphate shunt, glutathione stability decreased, and glycine incorporation into glutathione increased. However, in mild glucose-6-phosphate dehydrogenase deficiency with no clinical manifestation, glutathione stability and recycling via the hexose monophosphate shunt decreased and hexose monophosphate shunt activity decreased slightly. In glucosephosphate isomerase deficiency, more than half of glucose was metabolized via the hexose monophosphate shunt, while Embden-Meyerhof pathway activity and recycling via the hexose monophosphate shunt decreased. In a splenectomized patient with pyruvate kinase deficiency with reticulocytosis, high hexose monophosphate shunt activity, glucose consumption and lactate formation were observed; but in pyruvate kinase deficiency without splenectomy, decreased glutathione stability, low glucose consumption and lactate formation were observed.These results indicate that one abnormal enzyme may exert its influence on several enzyme systems and that it is valuable to investigate the metabolism of erythrocytes using this system in different enzymopathies as well as different variants of one enzyme deficiency.     

Fragility of abnormal erythrocytes evaluated by response to shear stress.

Shear stress is a potential cause of erythrocyte fragmentation and hemolysis in flowing blood. In this study, the response of abnormal human erythrocytes to shear stress in virto was evaluated using a concentric cylinder viscometer. Compared to normal red cells, deoxygenated erythrocytes from persons with sicle cell anemia were particularly susceptible to fragmentation and hemolysis by shear stress. Oxygenation of sicke cell blood improved the resistance of those red cells to shear stress; they remain, however, more susceptible to shear stress than normal erythrocytes. Erythrocytes from patients with iron deficiency, thalassemia minor, and erythrocyte pyruvate kinase deficiency showed fragmentation and hemolysis at threshold shear stresses intermediate between those ovserved for blood from patients with sickle cell anemia and normal persons. Blood samples from patients with hereditary spherocytosis were more resistant to shear stress than normal blood. These results indicate that there are important differences in the response of various red cells to shear stress.     

Evaluation of the efficacy of intravenous acetaminophen in the treatment of acute migraine attacks: a double-blind, placebo-controlled parallel group multicenter study

The efficacy of intravenous acetaminophen (1000mg) in the treatment of acute migraine attacks as an alternative to parenteral application of lysine acetylsalicylate or triptans was investigated, using a multi-center, randomized, double-blind, placebo controlled study design. Migraine diagnosis was made according to the International Headache Society Classification. Sixty patients were included in three headache outpatient centers (Neurology Departments of the Universities of Regensburg, Münster and München). In the acute migraine attack patients were treated intravenously with either 1000mg paracetamol (acetaminophen) or placebo. The primary end point was pain-free after 2h. Secondary efficacy criteria were pain-free after 24h or pain relief after 2hours and after 24hours. With regard to the efficacy criteria, 37% of patients reported pain relief or painfree after two hours, 12 patients after treatment with acetaminophen and 10 patients after treatment with placebo. Out of these, 3 patients in the acetaminophen and 4 patients in the placebo group were painfree. After 24hours 86% of the patients reported pain relief: 24 treated with acetaminophen and 27 treated with placebo. The results indicate, that 1000mg intravenous acetaminophen is not superior to placebo in treating severe acute migraine attacks.     

Multicellular origin of fibrosarcomas in mice induced by the chemical carcinogen 3-methylcholanthrene

The cellular origin of tumors induced by the chemical carcinogen 3-methylcholanthrene (MCA) was studied in mice with X-chromosome inactivation mosaicism. Because only one of the two X-chromosomes is active in XX somatic cells, a female heterozygous at the X-linked phosphoglycerate kinase (PGK-1) locus for the usual Pgk-1b gene and the variant Pgk-1a has two populations of cells, in the cells of one population, Pgk-1b is active and B-type enzyme is synthesized, whereas in cells of the other population, A-type enzyme is produced. Both enzyme types are found in normal tissues from these mosaic mice. A tumor developing from a single cell exhibits only one of the two PGK enzyme types, whereas a tumor with a multicellular origin expresses both enzymes (i.e., it has a double-enzyme phenotype). Five fibrosarcomas developing at the site of injection of 0.2 or 2.0 mg of MCA were analyzed. 36 of 38 fragments from the five tumors had double-enzyme PGK phenotypes. One piece from each of two tumors showed a single-enzyme phenotype. Histological, cell culture, and cloning studies indicate that the double-enzyme phenotypes reflect the presence of both types of malignant cells and not admixture of normal with neoplastic elements in the specimens tested for PGK. The results suggest strongly that these fibrosarcomas have a multicellular origin.     

Vitamin B(1) status assessed by direct measurement of thiamin pyrophosphate in erythrocytes or whole blood by HPLC: comparison with erythrocyte transketolase activation assay

BACKGROUND: The concentration of thiamin diphosphate (TDP) in erythrocytes is a useful index of thiamin status. We describe an HPLC method for TDP and its results in patients at risk of thiamin deficiency. METHODS: We used reversed-phase HPLC with postcolumn derivatization with alkaline potassium ferricyanide and fluorescence detection. Samples were deproteinized and injected directly onto a C(18) column. TDP concentrations in erythrocytes were compared with those in whole blood. Reference intervals for erythrocyte TDP (n = 147; 79 males and 68 females; mean age, 54 years) and whole blood TDP (n = 124; 68 males and 56 females; mean age, 54 years) were determined in an apparently healthy population. We compared erythrocyte TDP with results of the erythrocyte transketolase activation test in 63 patients who were considered at risk of thiamin deficiency. RESULTS: The method was linear to at least 200 microgram/L. The between-run CV was <8%. The lower limit of quantification for both whole blood and packed erythrocytes was 300 pg on column with a detection limit of 130 pg on column. Recovery of TDP from blood samples was >90%. TDP in erythrocytes correlated strongly with that in whole blood (r = 0.97). Reference intervals for erythrocyte and whole blood TDP were 280-590 ng/g hemoglobin and 275-675 ng/g hemoglobin, respectively. Of the 63 patients suspected of thiamin deficiency, 46 were normal by both TDP and activation tests, 13 were deficient by both tests, 1 was deficient by the activation test but had normal erythrocyte TDP concentrations, and 4 were normal by the activation test but had low TDP. CONCLUSIONS: The HPLC method is precise and yields results similar to the erythrocyte activation assay.