Deficiencias en piruvato quinasa y anemias hemolíticas / Pyruvate kinase deficiencies and hemolytic anemias

La piruvato quinasa cataliza la transformación de fosfoenolpiruvato y ADP en piruvato y ATP. La enzima, que aparece en todas las células vivas, es clave en la ruta central del metabolismo de carbohidratos. La deficiencia en piruvato quinasa, debida a una mutación en el gen PK-LR, origina alteraciones únicamente en los eritrocitos, porque estas células no son capaces de compensar el defecto enzimático. Por ello, la deficiencia de esta enzima es causa principal de la anemia hemolítica no esferocítica que puede provocar incluso la muerte de los pacientes. Las dificultades en la caracterización bioquímica de la PK nos ha llevado a estudiar la enzimopatía mediante técnicas de Biología Molecular. El trabajo se realizó sobre 10 pacientes con deficiencia en piruvato quinasa eritrocitaria. Mediante análisis molecular se han encontrado 11 mutaciones diferentes en los 17 alelos mutados: tres de estas mutaciones, G694A, A1150G y G1154A, no han sido previamente descritas. A las mutaciones que originan fuertes modificaciones en la estructura local de la molécula, observadas mediante estudios de modelización molecular, como consecuencia de un desajuste en el balance de las cargas eléctricas o por impedimento estérico, corresponden valores disminuidos de la actividad de la enzima en aquellos pacientes portadores de dichas mutaciones (AU)

Effects of fasting and glucocorticoids on hepatic gluconeogenesis assessed using two independent methods in vivo.

The purpose of this study was to compare the assessment of gluconeogenesis (GNG) in the overnight- and prolonged-fasted states and during chronic hypercortisolemia using the arteriovenous difference and [14C]phosphoenolpyruvate-liver biopsy techniques as well as a combination of the two. Two weeks before a study, catheters and flow probes were implanted in the hepatic and portal veins and femoral artery of dogs. Animals were studied after an 18-h fast (n = 8), a 42- or 66-h fast (n = 7), and an 18-h fast plus a continuous infusion of cortisol (3.0 microg. kg(-1). min(-1)) for 72 h (n = 7). Each experiment consisted of an 80-min tracer ([3-(3)H]glucose and [U-(14)C]alanine) and dye equilibration period (-80 to 0 min) and a 45-min sampling period. In the cortisol-treated group, plasma cortisol increased fivefold. In the overnight-fasted group, total GNG flux rate (GNG(flux)), conversion of glucose 6-phosphate to glucose (GNG(G-6-P-->Glc)), glucose cycling, and maximal GNG flux rate (GNG(max)) were 0.95 +/- 0.14, 0.65 +/- 0.06, 0.62 +/- 0.06, and 0.70 +/- 0.09 mg. kg(-1). min(-1), respectively. In the prolonged-fasted group, they were 1.50 +/- 0.18, 1.18 +/- 0.13, 0.40 +/- 0.07, and 1.28 +/- 0.10 mg. kg(-1). min(-1), whereas in the cortisol-treated group they were 1.64 +/- 0.33, 0.99 +/- 0.29, 1.32 +/- 0.24, and 0.91 +/- 0.13 mg. kg(-1). min(-1). These results demonstrate that GNG(G-6-P-->Glc) and GNG(max) were almost identical. However, these rates were 15-38% lower than GNG(flux) generated by a combination of the two methods. This difference was most apparent in the steroid-treated group, where the combination of the two methods (GNG(flux)) detected a significant increase in gluconeogenic flux.

Clinical application of phosphoenolpyruvate (PEP) to autologous transfusion of patients with open heart surgery.

Patients undergoing surgery for cardiac valve replacement were autologously transfused after their cryopreserved blood was treated with phosphoenolpyruvate. Five patients received red cells treated on the day of operation with a solution containing phosphoenolpyruvate, the other 6 serving as a control group. None of the patients received homologous blood. The treated red cells had a normal adenosine triphosphate concentration; in the control group a 10% decrease was noted. The 2,3-bisphosphoglycerate concentration was 211% of normal in red cells of the treated group and 69% of normal in the control group. The P50 values (mmHg) were 31.1 +/- 3.5 (treated), 20.3 +/- 1.7 (untreated), and 26.1 +/- 0.6 (fresh, not cryopreserved), respectively. Both the 2,3-bisphosphoglycerate and P50 values of the circulating blood were significantly (p < 0.02) increased in the patients receiving treated red cells. The increase in the 2,3-bisphosphoglycerate and P50 remained for 6 hours after transfusion at which time the levels of the 2,3-bisphosphoglycerate were 14.5 +/- 2.2 mumol/gHb (treated group) and 10.5 +/- 0.8 mumol/gHb (control group). Those of P50 were 28.1 +/- 1.5 mmHg (treated group) and 25.9 +/- 0.9 mmHg (control group). The adenosine triphosphate levels were not significantly different. The postoperative values of pH and hematocrit did not differ between the two groups. It was estimated that the oxygen delivery capacity in the circulating blood was about 30% higher in the patients receiving phosphoenolpyruvate treated blood than in those receiving untreated blood.

[Erythrocyte membrane and hereditary spherocytosis].

Hereditary spherocytosis (HS) is the most common hemolytic anemia caused by intrinsic defects of the erythrocyte membrane. Recent investigations have been directed toward the cytoskeleton of affected cells, and in some patients, the following several molecular lesions have been identified: (1) an abnormal spectrin incapable of binding to band 4.1 in dominantly inherited HS, (2) a deficiency of spectrin in severe, recessively inherited HS, (3) a deficiency of ankyrin and spectrin in severe, atypical HS, and (4) complete or partial defect of band 4.2 in some Japanese cases with HS. However, in the majority of patients with HS, the molecular lesion still remains unclear. We determined the transport rate of phosphoenolpyruvate (PEP) in HS erythrocytes, which was found to be specifically lower by almost half of the rate in erythrocytes from normal subjects or patients with other anemias. Since PEP is transported via band 3, the phenomenon may be a reflection of either structural abnormality of band 3 or dysfunction of band 3 caused by other primary lesions. Further investigations is needed to elucidate the underlying molecular defects in majority of patients with HS.

Site-directed mutagenesis in the effector site of Escherichia coli phosphofructokinase.

A new vector for the expression of phosphofructokinase (pfk-1) was constructed with pEMBL, which allows reliable, inducible, high-expression, and facile mutagenesis of the gene. Two mutants in the effector site of the enzyme were produced by site-specific mutagenesis of residue Tyr-55 to assess the role of its side chain in binding an allosteric inhibitor, phosphoenolpyruvate (PEP), and an activator, guanosine 5'-diphosphate (GDP): Tyr-55----Phe-55 and Try-55----Gly-55. The dissociation constant of PEP from the T state is unaffected by the mutations. Mutation of Tyr-55----Phe-55 only slightly increases the dissociation constant of GDP from the R state, indicating a minimal involvement of the hydroxyl group in binding. A 5.5-fold increase in the dissociation constant of GDP on the mutation of Tyr-55----Gly-55 suggests a small hydrophobic interaction of the aromatic ring of the tyrosine residue with guanine of GDP.