Erythrocyte Pyruvate Kinase Activity Research Articles

Mitapivat: A Quinolone Sulfonamide to Manage Hemolytic Anemia in Adults With Pyruvate Kinase Deficiency.

Pyruvate kinase (PK) deficiency is a rare enzyme-linked glycolytic defect resulting in mild-to-severe chronic persistent erythrocyte hemolysis. The disease is an autosomal recessive trait caused by mutations in the PK liver and red blood cell gene characterized by insufficient erythrocyte PK activity. PK deficiency is most diagnosed in persons of northern European descent and managed with packed red blood cell transfusions, chelation, and splenectomy with cholecystectomy. Mitapivat is the first approved therapy indicated for hemolytic anemia in adults with PK deficiency with the potential for delaying splenectomy in mild-moderate disease. Mitapivat is a PK activator that acts by allosterically binding to the PK tetramer and increases PK activity. The red blood cell form of PK is mutated in PK deficiency, which leads to reduced adenosine triphosphate, shortened red blood cell lifespan, and chronic hemolysis. The half-life of elimination is 3-5 hours, with 73% bioavailability, 98% plasma protein binding, and a median duration of response of 7 months. Mitapivat has been investigated through various clinical trials for different therapeutic indications. Pivotal trials that serve the primary focus throughout this article are ACTIVATE, ACTIVATE-T, and RISE. ACTIVATE is a phase 3, randomized, double-blind, placebo-controlled study that evaluated the efficacy and safety of mitapivat in adult patients who were not receiving regular blood transfusions. Contrarily, ACTIVATE-T explored the safety and efficacy of mitapivat in adults with PK deficiency who received regular blood transfusions. Both trials demonstrated favorable use of mitapivat in PK deficiency. Focusing on another indication, the ongoing RISE trial investigates the optimal dosage of mitapivat in sickle cell disease. Mitapivat is an appropriate treatment for adults with PK deficiency requiring transfusions and may be considered for patients with symptomatic anemia who do not require transfusions and/or PK deficiency with compensated hemolysis without overt anemia.

CARBOHYDRATES METABOLISM IN THE BLOOD OF RATS WITH IMPAIRED GLUCOSE TOLERANCE UNDER LONG TERM MELATONIN INJECTIONS.

The aim: To determine the influence of melatonin on the glucose level and content of malondialdehyde, activities of pyruvate kinase and glucose-6-phosphate dehydrogenase enzymes in the blood; histochemical features of glycogen distribution in liver of rats with impaired glucose tolerance. Materials and methods: Diabetes in rats was induced by intra-abdominal injection of a 5% solution of alloxan monohydrate at the rate of 170 mg/kg of body weight. Four days after animals were divided into rats with impaired glucose tolerance and melatonin-group with impaired glucose tolerance (5 mg/ kg «Sigma» USA, daily and intraperitoneal for 42 days starting from 5th day). Impaired glucose tolerance was determined by measurement of glucose profiles - fasting <5.6 mmol/l; postprandial (2h post-load) 7.8 - 11.0 mmol/l. Histochemical examination of the liver was performed according to the standard method of PAS-reaction staining. Statistical analysis was performed using Statistica 10 StatSoft Inc. Results: Pyruvate kinase activity in erythrocytes and optical density of glycogen in hepatocytes of animals with impaired glucose tolerance decreased on 18% and 11%, activity of glucose-6-phosphate dehydrogenase and content of malondialdehyde increased on 35% and 23%, respectively compared with the control. We have reached the recovery of the pyruvate kinase and normalization of glucose-6-phosphate dehydrogenase activities, malondialdehyde levels, glucose profiles in the blood as well as glycogen distribution in the liver caused by melatonin injections. Conclusions: We have determined that long term melatonin injections did better glucose tolerance in rats.

INFLUENCE OF MELATONIN ON AGE-RELATED CHANGES IN CARBOHYDRATE METABOLISM AND ANTIOXIDANT CAPACITY IN THE BLOOD OF ALLOXAN DIABETIC RATS

Melatonin and its metabolites have potent antioxidant/anti-inflammatory properties, and they have proven to be highly effective in a variety of disorders linked to inflammation and oxidative stress. The object of this experimental research was to ascertain the influence of aging on the level of basal glycemia and activities of glucose-6-phosphate dehydrogenase [EC1.1.1.49], pyruvate kinase [EC 2.7.1.40] and glutathione reductase [EC1.6.4.2] in erythrocytes of alloxan diabetic rats on the background of melatonin injections. Methods: We used 100 male Wistar rats, two age groups: the - 2-month (adult), and II - 4-month (old). Alloxan diabetes was evoked via injecting the rats with a 5% solution of alloxan monohydrate intraperitoneally in a dose of 170 mg/kg. Four days after diabetes induction, rats were divided into diabetic (untreated) and melatonin-diabetic group (10 mg/kg, daily and intraperitoneally for six weeks). Blood was taken from the tail vein evaluate the basal glycemia on 5-th and 47-th day after the injection of alloxan. Rats were sacrificed at the 47-th day of the experiment accordance with the ethical treatment of animals. Determinations of the enzymes activities were by standard methods. Statistical analysis was performed using Statistica 10 StatSoft Inc. Results. The level of basal glycemia on the fifth day of the experiment in animals of both groups increased on average by 115% from baseline values. We founded that on 47-th day this index was higher in group of old rats on 20% more than in adult rats. Pyruvate kinase activity in erythrocytes of adult and old animals with diabetes decreased by 34% and 51% respectively compared with the control. glucose-6-phosphate dehydrogenase activity in erythrocytes of adult and old animals with diabetes decreased by 25% and 44% respectively compared with the control on 47-th day. The changes may be the result of age-related disorders of glucose metabolism due to disturbances in free radical mechanisms. Glutathione reductase activity in erythrocytes of adult and old animals with diabetes decreased by 30% and 36% respectively compared with the control on 47-th day. A 42-days injection of melatonin to the alloxan diabetic rats of both groups contributed to a normalization of the level of basal glycemia, the activities of pyruvate kinase and glutathione reductase in the rat blood, as well as to a considerable increase of the activity of glucose-6-phosphate dehydrogenase, whose level exceeded by average 9% this particular index in the control group of animals. Under the influence of melatonin increase activity of glucose-6-phosphate dehydrogenase in the blood of rats may be due to the increasing number of substrate for glucose-6-phosphate dehydrogenase (stimulating the flow of glucose into cells and its phosphorylation) and direct action. Conclusion. In this case melatonin probably increases use of glucose for regeneration of NADPH2 and aerobic oxidation of glucose that indicate an acceleration of antioxidative protection and energy production in blood of adult and old diabetic rats.

AGE-RELATED CHANGES OF GLYCOLYTIC ACTIVITY AND ANTIOXIDANT CAPACITY IN THE BLOOD OF ALLOXAN DIABETIC RATS

The increasing incidence of type 1 diabetes coupled with advances in treatment of type 1 diabetes has resulted in an unprecedented number of older adults living with and controllable type 1 diabetes. The objective of this experimental study was to assess the impact of aging on the level of basal glycaemia and activities of glucose-6-phosphate dehydrogenase [EC1.1.1.49], pyruvate kinase [EC 2.7.1.40] and glutathione reductase [EC1.6.4.2] in erythrocytes of alloxan-diabetic rats. Methods: We used 100 male Wistar rats, divided into two age groups: I group included- 2-month (adult) animals, and II group was made up of 4-month (old) animals. Diabetes was modelled by injecting the rats with 5% solution of alloxan monohydrate intraperitoneally in a dose of 170 mg/kg. Blood was taken from the tail vein to evaluate the basal glycaemia on 5-th and 47-th day after the alloxan injection. Rats were sacrificed on the 47-th day of the experiment in accordance with the regulations on ethical treatment of vertebrates. The assessment of the activity of the enzymes was carried out by standard methods. Statistical analysis was performed by using Statistica 10 StatSoft Inc. Results. The level of basal glycaemia on the fifth day of the experiment in the animals of both groups went up on average by 115% from baseline values. We founded that on 47-th day this index was higher in group of old rats by 20% than in adult rats. Pyruvate kinase activity in erythrocytes of adult and old animals with diabetes decreased by 35% and 50% respectively compared with the control. Glucose-6-phosphate dehydrogenase activity in erythrocytes of adult and old animals with diabetes decreased by 27% and 45% respectively compared with the control on 47-th day. The changes may be considered as the result of age-related disorders of glucose metabolism due to disturbances in free radical mechanisms. Glutathione reductase activity in erythrocytes of adult and old animals with diabetes decreased by 29% and 35% respectively compared with the control on 47-th day. Conclusion. We have determined when getting aged, the alloxan-diabetic rats demonstrate changes in the sensitivity of pyruvate kinase, glucose-6-phosphate dehydrogenase and glutathione reductase activities in erythrocytes resulted from the effect of diabetes mellitus factors (hyperglycaemia). We can suggest that glycaemic control is key purpose for older patients with type 1 diabetes in order to prevent of complication, which can be aggravated with age.

Hereditary spherocytosis is associated with decreased pyruvate kinase activity due to impaired structural integrity of the red blood cell membrane.

Hereditary spherocytosis (HS) is characterised by increased osmotic fragility and enhanced membrane loss of red blood cells (RBC) due to defective membrane protein complexes. In our diagnostic laboratory, we observed that pyruvate kinase (PK) activity in HS was merely slightly elevated with respect to the amount of reticulocytosis. In order to evaluate whether impaired PK activity is a feature of HS, we retrospectively analysed laboratory data sets from 172 unrelated patients with HS, hereditary elliptocytosis (HE), glucose-6-phosphate dehydrogenase (G6PD) or PK deficiency, sickle cell or haemoglobin C disease, or β-thalassaemia minor. Results from linear regression analysis provided proof that PK activity decreases with rising reticulocyte counts in HS (R2 =0·15; slope=9·09) and, less significantly, in HE (R2 =0·021; slope=8·92) when compared with other haemolytic disorders (R2 ≥0·65; slopes≥78·6). Reticulocyte-adjusted erythrocyte PK activity levels were significantly lower in HS and even declined with increasing reticulocytes (R2 =0·48; slope=-9·74). In this report, we describe a novel association between HS and decreased PK activity that is apparently caused by loss of membrane-bound PK due to impaired structural integrity of the RBC membrane and may aggravate severity of haemolysis in HS.

Phenotypic and molecular genetic analysis of Pyruvate Kinase deficiency in a Tunisian family

Pyruvate Kinase (PK) deficiency is the most frequent red cell enzymatic defect responsible for hereditary non-spherocytic hemolytic anemia. The disease has been studied in several ethnic groups. However, it is yet an unknown pathology in Tunisia. We report here, the phenotypic and molecular investigation of PK deficiency in a Tunisian family.This study was carried out on two Tunisian brothers and members of their family. Hematological, biochemical analysis and erythrocyte PK activity were performed. The molecular characterization was carried out by gene sequencing technique.The first patient died few hours after birth by hydrops fetalis, the second one presented with neonatal jaundice and severe anemia necessitating urgent blood transfusion. This severe clinical picture is the result of a homozygous mutation of PKLR gene at exon 8 (c.1079G>A; p.Cys360Tyr).Certainly, this research allowed us to correlate the clinical phenotype severity with the identified mutation. Moreover, this will help in understanding the etiology of unknown anemia in our country.

Decrease of aldolase and pyruvate kinase activity in erythrocytes of individuals with male hypogonadism as an expression of lack of androgen influence on the bone marrow.

The authors have demonstrated the statistically significant decrease of aldolase and pyruvate kinase activity in erythrocytes in a group of men with hypogonadism and with a significant defect of gonad endocrinologic function. A similar phenomenon appeared in erythrocytes in a group of sexually mature rabbits after castration. In the course of substitutional treatment with testosterone esters given intramuscularly, the increase of activity characteristic for both examined glycolytic enzymes was observed in both experimental groups, i.e. the patients and the animals. The pyruvate kinase increase, however, was not statistically significant in the examined group of men, which resulted from the small size of the group. The authors suggest the existence of testosterone influence on regular activity of bone marrow glycolytic enzymes, and consequently on the mature erythrocytes. The decrease of aldolase and pyruvate kinase activity in erythrocytes of patients with male hypogonadism may decrease the erythrocytes valence. This ist another biochemical argument for the need of conducting long-term substitutional therapy in cases of male hypogonadism.

Erythrocyte pyruvate kinase deficiency: relations of residual enzyme activity, altered regulation of defective enzymes and concentrations of high-energy phosphates with the severity of clinical manifestation.

The defective enzymes of 54 patients with pyruvate kinase (PK) deficiency were characterized according to the recommendations of the International Committee for Standardization in Haematology (ICSH). The erythrocyte PK activity in whole blood was calculated considering the 16-fold higher activity of the reticulocyte enzyme (AR) compared to the erythrocyte enzyme (AE). The following parameters turned out to give a good correlation to the degree of haemolytic anaemia and can therefore serve as a prognostic tool: All patients with a severe course of the disease had residual erythrocyte PK activities less than 33% of the normal enzymes (percentage activity), and patients with mild haemolytic anaemia exhibited residual activity values below and above this threshold value. Studies of enzyme cooperative showed that positive cooperative or mixed cooperative phosphoenolpyruvate (PEP) binding with a predominant positive cooperative part appeared in all cases with a mild clinical course, and about one-third of the severe ones. Negative cooperativity or mixed cooperativity with predominant negative cooperative part was observed only with severe haemolytic anaemia. Furthermore, the determination of glucose-6-phosphate (G-6-P) turned out to be a good prognostic criterion, i.e. all patients with mild clinical course exhibited G-6-P-concentrations lower than 0.11 mumol/l red blood cells. In the case of patients with severe haemolytic anaemia, about 80% showed values higher than 0.11 mumol/l RBC.

Erythrocyte Pyruvate Kinase Activity in Term and Preterm Infants

The activity of erythrocyte pyruvate kinase (PK), one of the most important enzymes in the anaerobic glycolytic pathway, was measured in a total of 57 healthy (22 term and 35 preterm) infants. The mean PK activity was 8.98 ± 3.43 IU/gHb in term and 16.56 ± 7.26 IU/gHb in preterm infants. The mean PK activity was significantly higher in preterm babies than term infants (16.56 ± 7.26 IU/gHb and 8.98 ± 3.43 IU/gHb, respectively) (P <. 001). A significant negative correlation was found between gestational age, birth weight, and PK activity (r = 0.40, P <. 05; r = -0.37, P <. 05). No correlation was found between postnatal age and PK activity in both preterm and term infants. The increased PK activity in preterm babies was thought to be due to increased glycolytic activity and energy production in these infants.

Hereditary Hemolytic Anemia Caused by Diverse Point Mutations of Pyruvate Kinase Gene Found in Japan and Hong Kong

We identified four distinct point mutations in homozygous pyruvate kinase (PK) variants in Japanese and Chinese patients with chronic nonspherocytic hemolytic anemia. All gene abnormalities were missense mutations that caused single amino acid substitutions. 1261A (Q421K) and 1436A (R436H), which were identified in PK Sendai and PK Shinshu, had been found in unrelated Japanese and Amish PK variants, respectively. The clinical severity and extremely low residual erythrocyte PK activity of PK Shinshu as well as of the Amish PK might be caused partly by aberrant splicing, because the 1436A mutation changes a nucleotide at the last nucleotide in the exon 10. Recently, we diagnosed a 42-year-old Japanese woman with chronic nonspherocytic hemolytic anemia as having a homozygous PK deficiency. DNA sequencing of the variant PK gene showed a homozygous missense mutation at 1403GCT-->GTT, resulting in a single amino acid substitution from 468la-->Val. The gene mutation is likely to impair the allostericity of this enzyme, speculated from the tertiary structure. A homozygous missense mutation in PK Hong Kong, a boy of a non-Han southern Chinese minority group, was identified in exon 7 of the human L-PK gene, 941ATT-->ACT, resulting in a single amino acid substitution from 314lle-->Thr. The R-PK activity is expected to be severely affected, because the mutated amino acid residue is located between the 313 Lys and the 315 Glu, which are very important for acid-base catalysis and magnesium binding, respectively. Both the R- and M2-type PK were shown by polyacrylamide gel electrophoresis of the PK Hong Kong erythrocyte lysate, and this is the first report of a homozygous individual whose erythrocytes contain the immature (M2)-type isozyme.

Hereditary hemolytic anemia caused by diverse point mutations of pyruvate kinase gene found in Japan and Hong Kong

We identified four distinct point mutations in homozygous pyruvate kinase (PK) variants in Japanese and Chinese patients with chronic nonspherocytic hemolytic anemia. All gene abnormalities were missense mutations that caused single amino acid substitutions. 1261A (Q421K) and 1436A (R436H), which were identified in PK Sendai and PK Shinshu, had been found in unrelated Japanese and Amish PK variants, respectively. The clinical severity and extremely low residual erythrocyte PK activity of PK Shinshu as well as of the Amish PK might be caused partly by aberrant splicing, because the 1436A mutation changes a nucleotide at the last nucleotide in the exon 10. Recently, we diagnosed a 42-year-old Japanese woman with chronic nonspherocytic hemolytic anemia as having a homozygous PK deficiency. DNA sequencing of the variant PK gene showed a homozygous missense mutation at 1403GCT- -&gt;GTT, resulting in a single amino acid substitution from 468la--&gt;Val. The gene mutation is likely to impair the allostericity of this enzyme, speculated from the tertiary structure. A homozygous missense mutation in PK Hong Kong, a boy of a non-Han southern Chinese minority group, was identified in exon 7 of the human L-PK gene, 941ATT--&gt;ACT, resulting in a single amino acid substitution from 314lle--&gt;Thr. The R- PK activity is expected to be severely affected, because the mutated amino acid residue is located between the 313 Lys and the 315 Glu, which are very important for acid-base catalysis and magnesium binding, respectively. Both the R- and M2-type PK were shown by polyacrylamide gel electrophoresis of the PK Hong Kong erythrocyte lysate, and this is the first report of a homozygous individual whose erythrocytes contain the immature (M2)-type isozyme.

Rapid determination of erythrocyte pyruvate kinase activity

We report a new potentiometric method for determining pyruvate kinase (PK). Enzymatic activity is measured by monitoring the change in pH produced in the reaction buffer under International Committee for Standardization in Haematology (ICSH) standardized assay conditions, and the lactate dehydrogenase reaction is automatically subtracted in each measuring cycle. The analysis, performed at 37 degrees C, requires a 10-microL sample (isolated erythrocytes or whole blood) and is completed in 2.5 min. The intra-assay CV is < 4% (PK between 3 and 35 U/g Hb); the interassay CV is 4.0% (PK 15 U/g Hb); results are linear from 3 to 30 U/g Hb. A good correlation with the ICSH reference method (x) was found: y = 1.011x - 0.05; n = 32; r = 0.9939; Sylx = 0.75 (units: U/g Hb). The reference intervals of the PK activity in isolated erythrocytes (RBC-PK) were estimated in 89 normal subjects. We found that women possess a higher RBC-PK than do men (P < 0.0001) and that the biological variability (CVb) of RBC-PK is 13.5%. Applications of the proposed method to the hematological routine are reported.

Inherited erythrocyte pyruvate kinase deficiency in a beagle dog.

A 1-year-old, female Beagle dog with minimal exercise intolerance was found to have a persistent, severe, and highly regenerative anemia, splenomegaly, and progressive osteosclerosis. Despite near-normal in vitro erythrocyte pyruvate kinase (PK) activity, the authors diagnosed PK deficiency by demonstrating a glycolytic block at the PK step, the lack of normal R-type PK isoenzyme, and the presence of M(2)-type PK in the animal's erythrocytes. The dam had half-normal erythrocyte PK activity, which supports an autosomal recessive mode of inheritance. We conclude from our studies that close similarities exist between erythrocyte PK deficiency in Beagle, Basenji, and West Highland White Terrier dogs and that this form of PK deficiency may be more widespread than previously thought.

Inherited erythrocyte Syruvate kinase eficiency in the West Highland white terrier

ABSTRACTAn eight‐month‐old male West Highland white terrier (WHWT) presented with mild exercise intolerance and splenomegaly caused by a severe and highly regenerative haemolytic anaemia that persisted until the dog died at two years of age. There was marked erythroid hyperplasia in the bone marrow and progressive osteosclerosis. Erythrocyte pyruvate kinase (PK) deficiency was confirmed by demonstrating a block in glycolysis at the PK step, abnormal erythrocyte PK kinetics, absence of R‐type PK isoenzyme, and the presence of M2‐type PK not normally expressed in erythrocytes. From the study of family members it is concluded that erythrocyte PK deficiency in the WHWT is inherited as an autosomal recessive trait. Heterozygotes have about half‐normal erythrocyte PK activity, do not express the M2‐type PK isoenzyme and are asymptomatic. This disease closely resembles erythrocyte PK deficiency in the basenji breed and should be considered as a differential diagnosis in young dogs with severe haemolytic anaemia.

Ascorbate‐induced hyperoxalaemia has no significant effect on lactate generation or erythrocyte 2,3, diphosphoglycerate in dialysis patients

To examine the possible effects of hyperoxalaemia on anaerobic metabolism and erythrocyte pyruvate kinase activity, we induced a rise in plasma oxalate in 11 dialysis patients by the oral administration of ascorbic acid, 500 mg day-1 for 3 weeks. Blood samples were taken from the same antecubital vein before and after the supplementation period, without venous stasis, after an overnight fast. This protocol allowed patients to be used as their own controls. Five healthy subjects underwent an identical protocol to exclude any effect of ascorbate per se. Mean (SEM) plasma oxalate (mumol l-1) rose from 30.3 (3.5) to 48.4 (6.1) in patients and from 1.4 (0.2) to 6.8 (0.9) in healthy subjects. Whole blood ascorbate (mg l-1) rose from 7.0 (0.7) to 26.6 (2.5) in patients and from 9.3 (1.2) to 17.8 (1.8) in healthy subjects (reference range 7.5-20.0 mg l-1). No changes were observed in either group in plasma creatinine, bicarbonate, haemoglobin, or erythrocyte 2,3,diphosphoglycerate (2,3 DPG) after the 3 week supplementation period. Before supplementation lactate generation (area under curve, mmol min l-1) in the 5 min following a 60 s period of standardized ischaemic forearm exercise was significantly (P = 0.026) greater in patients [69.1 (4.7)] than in healthy subjects [46.9 (6.7)]; no significant change in lactate generation occurred in either group after ascorbate-induced hyperoxalaemia. We conclude that changes in plasma oxalate of the order of 20 mumol l-1 have no significant effect on lactate generation or 2,3,DPG levels in uraemic subjects.

Seasonal variations in the responses of glycolytic intermediates of human erythrocytes to acute cold exposure.

Seven male students were studied to observe the effects of acute cold exposure (at 10°C for 60 min) on erythrocyte concentrations of glycolytic intermediates in summer and in winter. The subjects shivered slightly but frankly in both experiments. Significant decreases were observed in the concentrations of pyruvate and lactate during body cooling in summer, but not in winter. The lactate concentration remained significantly reduced 15 min after cold exposure. After 60 min of cold exposure in summer, a negative crossover point appeared to exist between phosphoenolpyruvate and pyruvate and erythrocyte pyruvate kinase activity showed a significant decrease. No seasonal difference was observed in the initial control values of the intermediates measured. From these results and the fact that glucose, pyruvate and lactate are evenly distributed between erythrocytes and plasma, it is likely that erythrocytes and skeletal muscles need less fuel substrate, glucose during cold exposure in winter than in summer, suggesting that an increased economy of energy for homeostasis is achieved.

Enrichment of blood units with young red cells (neocytes) with the IBM 2991 cell washer.

The transfusion of young red blood cells (neocytes) is a complementary approach to the present treatment of thalassaemia major patients. Fresh neocytes were harvested from 34 volunteers with the IBM 2991 cell washer (CW). The upper part of 24 units was separated into 6 aliquots (T1-T6) of 15 ml each and the residue homogenized. Four biological criteria were used to evaluate young red cell quality, the erythrocyte pyruvate kinase activity, the mean corpuscular volume, the reticulocyte count and ektacytometry; each sample (T1-T6) showed an enrichment with neocytes as compared with the standard unit. A real efficiency of this technique with the IBM 2991 CW was obtained with the first third of the unit, corresponding to the T1-T4 fractions. The estimation of the theoretical mean life span of T1-T2 was 270 days. The last 10 units were separated into two halves: the enrichment of the first half was worse than in the first third of the previous technique. We concluded that the transfusional program of thalassaemia patients could profit by the use of the most enriched part of a standard blood unit with the IBM 2991 CW.

Nonspherocytic haemolytic anaemia due to phosphofructokinase deficiency in an English Springer Spaniel

ABSTRACTA five‐year‐old female English Springer Spaniel was evaluated for chronic episodic anaemia, pigmenturia and icterus. There was increased erythrocyte pyruvate kinase activity, increased in vitro erythrocyte alkaline fragility, decreased erythrocyte 2,3‐diphosphoglycerate activity and severely decreased erythrocyte phosphofructokinase activity. The clinical features and haematological values were identical with erythrocyte phosphofructokinase deficiency and resulting nonspherocytic haemolytic anaemia recently described in this breed.

A new pyruvate kinase mutation with hyperactivity in the mouse.

A mouse mutant with pyruvate kinase (PK) hyperactivity has been found in offspring of 1-ethyl-1-nitrosourea (ENU)-treated male mice. The activity alteration was detected in the blood and could also be found in the liver but not in the muscle, kidney, heart, spleen, lung, or brain. Heterozygous mice have erythrocyte PK activity enhanced up to about 160% and homozygotes up to about 240%, compared to homozygous wild types. The mutation is codominantly expressed. The heterozygous and homozygous mutants are viable and fully fertile and do not show symptoms of erythrocytosis. The mutation does not affect the heat stability, the electrophoretic mobility, or the Km (for phosphoenolpyruvate) of the PK molecule. It is suggested that the regulatory locus of PK-1 is affected by this mutation. The observations support also the theory of one structural locus for the erythrocyte and liver isozymes.

Red cell pyruvate kinase deficiency: An optimised assay

As a result of using a variety of assay techniques, rather different values for the rate of the pyruvate kinase (PK) reaction were obtained. Kinetic measurements performed with red cell PK using the stopped-flow as well as the “classical” spectrophotometric method demonstrated that only the initiation of the reaction with ADP yields reproducible results in the standard assay. Moreover, the influence of substrate concentrations, activator concentrations, ionic strength, buffer system, temperature and duration of preincubation were thoroughly investigated and were shown to play an important role. As a consequence of our kinetic measurements, a modified optimised assay composition together with new normal values for erythrocyte PK activity in haemolysate are presented. For better characterisation and comparability of PK variants, we suggest the use of the optimised assay composition described and that the reaction be initiated with ADP.