Familial pseudohyperkalemia induces significantly higher levels of extracellular potassium in early storage of red cell concentrates without affecting other standard measures of quality: A case control and allele frequency study.

BACKGROUND: Familial pseudohyperkalemia (FP) is characterized by an increased rate of potassium leakage in refrigerated red cells and is associated with the minor allele of the single nucleotide polymorphism rs148211042 (R723Q) in the ABCB6 gene. The study aims were to obtain the minor allele frequencies of ABCB6 variants and to measure supernatant potassium accumulation, and other red cell storage parameters, in red cell concentrates (RCC) from carriers of variant rs148211042 under standard blood bank conditions. STUDY DESIGN: Whole blood units were collected from 6 FP individuals and 11 controls and processed into RCC in additive solution. RCC were sampled and tested over cold storage for full blood count, extracellular potassium, glucose, lactate, microvesicle release, deformability, hemolysis, pH, adenosine triphosphate, and 2,3-diphosphoglycerate. RESULTS: Screening of genotyped cohorts identified that variant rs148211042 is present in 1 in 394 British citizens of European ancestry. FP RCC had significantly higher supernatant potassium at all time points from day 3 onwards (p < .001) and higher mean cell volume (p = .032) than controls. The initial rate of potassium release was higher in FP RCC; supernatant potassium reached 46.0 (23.8-57.6) mmol/L (mean [range]) by day 5, increasing to 68.9 (58.8-73.7) mmol/L by day 35. Other quality parameters were not significantly different between FP RCC and controls. CONCLUSION: These data suggest that if a blood donor has FP, reducing the RCC shelf-life to 5 days may be insufficient to reduce the risk of hyperkalemia in clinical scenarios such as neonatal large volume transfusion.

New insights on hereditary erythrocyte membrane defects.

After the first proposed model of the red blood cell membrane skeleton 36 years ago, several additional proteins have been discovered during the intervening years, and their relationship with the pathogenesis of the related disorders have been somewhat defined. The knowledge of erythrocyte membrane structure is important because it represents the model for spectrin-based membrane skeletons in all cells and because defects in its structure underlie multiple hemolytic anemias. This review summarizes the main features of erythrocyte membrane disorders, dividing them into structural and altered permeability defects, focusing particularly on the most recent advances. New proteins involved in alterations of the red blood cell membrane permeability were recently described. The mechanoreceptor PIEZO1 is the largest ion channel identified to date, the fundamental regulator of erythrocyte volume homeostasis. Missense, gain-of-function mutations in the PIEZO1 gene have been identified in several families as causative of dehydrated hereditary stomatocytosis or xerocytosis. Similarly, the KCNN4 gene, codifying the so called Gardos channel, has been recently identified as a second causative gene of hereditary xerocytosis. Finally, ABCB6 missense mutations were identified in different pedigrees of familial pseudohyperkalemia. New genomic technologies have improved the quality and reduced the time of diagnosis of these diseases. Moreover, they are essential for the identification of the new causative genes. However, many questions remain to solve, and are currently objects of intensive studies.

Functional characterization of novel ABCB6 mutations and their clinical implications in familial pseudohyperkalemia.

Isolated familial pseudohyperkalemia is a dominant red cell trait characterized by cold-induced 'passive leak' of red cell potassium ions into plasma. The causative gene of this condition is ABCB6, which encodes an erythrocyte membrane ABC transporter protein bearing the Langereis blood group antigen system. In this study analyzing three new families, we report the first functional characterization of ABCB6 mutants, including the homozygous mutation V454A, heterozygous mutation R276W, and compound heterozygous mutations R276W and R723Q (in trans). All these mutations are annotated in public databases, suggesting that familial pseudohyperkalemia could be common in the general population. Indeed, we identified variant R276W in one of 327 random blood donors (0.3%). Four weeks' storage of heterozygous R276W blood cells resulted in massive loss of potassium compared to that from healthy control red blood cells. Moreover, measurement of cation flux demonstrated greater loss of potassium or rubidium ions from HEK-293 cells expressing ABCB6 mutants than from cells expressing wild-type ABCB6. The R276W/R723Q mutations elicited greater cellular potassium ion efflux than did the other mutants tested. In conclusion, ABCB6 missense mutations in red blood cells from subjects with familial pseudohyperkalemia show elevated potassium ion efflux. The prevalence of such individuals in the blood donor population is moderate. The fact that storage of blood from these subjects leads to significantly increased levels of potassium in the plasma could have serious clinical implications for neonates and infants receiving large-volume transfusions of whole blood. Genetic tests for familial pseudohyperkalemia could be added to blood donor pre-screening. Further study of ABCB6 function and trafficking could be informative for the study of other pathologies of red blood cell hydration.

Missense mutations in the ABCB6 transporter cause dominant familial pseudohyperkalemia.

Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by increased serum [K(+)] in whole blood stored at or below room temperature, without additional hematological abnormalities. Functional gene mapping and sequencing analysis of the candidate genes within the 2q35-q36 critical interval identified-in 20 affected individuals among three multigenerational FP families-two novel heterozygous missense mutations in the ABCB6 gene that cosegregated with disease phenotype. The two genomic substitutions altered two adjacent nucleotides within codon 375 of ABCB6, a porphyrin transporter that, in erythrocyte membranes, bears the Langereis blood group antigen system. The ABCB6 R375Q mutation did not alter the levels of mRNA or protein, or protein localization in mature erythrocytes or erythroid precursor cells, but it is predicted to modestly alter protein structure. ABCB6 mRNA and protein levels increase during in vitro erythroid differentiation of CD34(+) erythroid precursors and the erythroleukemia cell lines HEL and K562. These data suggest that the two missense mutations in residue 375 of the ABCB6 polypeptide found in affected individuals of families with chromosome 2-linked FP could contribute to the red cell K(+) leak characteristic of this condition.

Human RhAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells.

In red cells, Rh-associated glycoprotein (RhAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pH(i)) changes. Recently, overhydrated hereditary stomatocytosis (OHSt), a rare dominantly inherited hemolytic anemia, was found to be associated with a mutation (Phe65Ser or Ile61Arg) in RHAG. Ghosts from the erythrocytes of four of the OHSt patients with a Phe65Ser mutation were resealed with a pH-sensitive probe and submitted to ammonium gradients. Alkalinization rate constants, reflecting NH(3) transport through the channel and NH(3) diffusion unmediated by RhAG, were deduced from time courses of fluorescence changes. After subtraction of the constant value found for Rh(null) lacking RhAG, we observed that alkalinization rate constant values decreased ∼50% in OHSt compared with those of controls. Similar RhAG expression levels were found in control and OHSt. Since half of the expressed RhAG in OHSt most probably corresponds to the mutated form of RhAG, as expected from the OHSt heterozygous status, this dramatic decrease can be therefore related to the loss of function of the Phe65Ser-mutated RhAG monomer.

Label-free oxygen-metabolic photoacoustic microscopy in vivo.

Almost all diseases, especially cancer and diabetes, manifest abnormal oxygen metabolism. Accurately measuring the metabolic rate of oxygen (MRO(2)) can be helpful for fundamental pathophysiological studies, and even early diagnosis and treatment of disease. Current techniques either lack high resolution or rely on exogenous contrast. Here, we propose label-free metabolic photoacoustic microscopy (mPAM) with small vessel resolution to noninvasively quantify MRO(2) in vivo in absolute units. mPAM is the unique modality for simultaneously imaging all five anatomical, chemical, and fluid-dynamic parameters required for such quantification: tissue volume, vessel cross-section, concentration of hemoglobin, oxygen saturation of hemoglobin, and blood flow speed. Hyperthermia, cryotherapy, melanoma, and glioblastoma were longitudinally imaged in vivo. Counterintuitively, increased MRO(2) does not necessarily cause hypoxia or increase oxygen extraction. In fact, early-stage cancer was found to be hyperoxic despite hypermetabolism.

Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S.

Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li(+) and (86)Rb(+), with secondarily increased (86)Rb(+) influx sensitive to ouabain and to bumetanide. Increased RhAG-associated (14)C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li(+), (86)Rb(+), and (14)C-MA were pharmacologically distinct, and Li(+) uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH(4)(+) and Gd(3+). RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH(3)/NH(4)(+), but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA(+)). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH(4)Cl, but MA/MA(+) elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li(+) substitution or bath addition of 5 mM NH(4)Cl or MA/MA(+). These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH(3)/NH(4)(+) and MA/MA(+); 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA(+) transport, and decreased NH(3)/NH(4)(+)-associated depolarization; and 3) RhAG transports NH(3)/NH(4)(+) and MA/MA(+) by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms.

Stomatin-deficient cryohydrocytosis results from mutations in SLC2A1: a novel form of GLUT1 deficiency syndrome.

The hereditary stomatocytoses are a series of dominantly inherited hemolytic anemias in which the permeability of the erythrocyte membrane to monovalent cations is pathologically increased. The causative mutations for some forms of hereditary stomatocytosis have been found in the transporter protein genes, RHAG and SLC4A1. Glucose transporter 1 (glut1) deficiency syndromes (glut1DSs) result from mutations in SLC2A1, encoding glut1. Glut1 is the main glucose transporter in the mammalian blood-brain barrier, and glut1DSs are manifested by an array of neurologic symptoms. We have previously reported 2 cases of stomatin-deficient cryohydrocytosis (sdCHC), a rare form of stomatocytosis associated with a cold-induced cation leak, hemolytic anemia, and hepatosplenomegaly but also with cataracts, seizures, mental retardation, and movement disorder. We now show that sdCHC is associated with mutations in SLC2A1 that cause both loss of glucose transport and a cation leak, as shown by expression studies in Xenopus oocytes. On the basis of a 3-dimensional model of glut1, we propose potential mechanisms underlying the phenotypes of the 2 mutations found. We investigated the loss of stomatin during erythropoiesis and find this occurs during reticulocyte maturation and involves endocytosis. The molecular basis of the glut1DS, paroxysmal exercise-induced dyskinesia, and sdCHC phenotypes are compared and discussed.

South-east Asian ovalocytosis and the cryohydrocytosis form of hereditary stomatocytosis show virtually indistinguishable cation permeability defects.

The hereditary stomatocytoses are a group of dominantly inherited conditions in which the osmotic stability of the red cell is compromised by abnormally high cation permeability. This report demonstrates the very marked similarities between the cryohydrocytosis form of hereditary stomatocytosis and the common tropical condition south-east Asian ovalocytosis (SAO). We report two patients, one showing a novel cryohydrocytosis variant (Ser762Arg in SLC4A1) and a case of SAO. Both cases showed a mild haemolytic state with some stomatocytes on the blood film, abnormal intracellular sodium and potassium levels which were made markedly abnormal by storage of blood at 0°C, increased cation 'leak' fluxes at 37°C and increased Na(+) K(+) pump activity. In both cases, the anion exchange function of the mutant band 3 was destroyed. Extensive electrophysiological studies comparing the cation leak and conductance in Xenopus laevis oocytes expressing the two mutant genes showed identical patterns of abnormality. These data are consistent with the cryohydrocytosis form of hereditary stomatocytosis and we conclude that the cation leak in SAO is indistinguishable from that in cryohydrocytosis, and that SAO should be considered to be an example of hereditary stomatocytosis.

Adrenal insufficiency and abnormal genitalia in a 46XX female with Smith-Lemli-Opitz syndrome.

OBJECTIVE: To report the first case of a 46XX female infant with Smith-Lemli-Opitz syndrome (SLOS), adrenal insufficiency and abnormal genitalia. METHODS: The patient was assessed for hormonal status on day 4 and 6 months later and was followed-up from the study time (2.5 years of age). RESULTS: The patient had a persistent urogenital sinus, posterior labial fusion without clitoromegaly. She presented with a salt-wasting syndrome on day 4. Adrenal insufficiency was confirmed. Adrenal androgen levels, including 17-hydroxyprogesterone and 11-deoxycortisol were moderately elevated. CONCLUSION: Children with SLOS should be assessed for adrenal insufficiency. In female infants, abnormal external genitalia can be observed even if the precise mechanism behind these abnormalities is yet to be determined.

Glucose-insulin infusion for the early treatment of non-oliguric hyperkalemia in extremely-low-birth-weight infants.

In order to investigate the status of non-oliguric hyperkalemia and to evaluate glucose-insulin infusion treatment among extremely-low-birth-weight (ELBW) infants, 161 infants weighting less than 1000 gm at birth were enrolled for this study. They were divided into two groups: a hyperkalemic group and a non-hyperkalemic group. Hyperkalemia was defined here as a serum potassium level of greater than 6 mEq/L in a non-hemolyzed arterial blood sample. A glucose-insulin infusion was administered to the patients when hyperkalemia was detected in them during the first few days after birth. The infusion was discontinued when the serum potassium levels had been less than 6 mEq/L and stabilized for 6 hours. The incidence of non-oliguric hyperkalemia among ELBW infants in this study was 58% (93/161). The mean gestational age of neonates was 25.7 +/- 1.8 weeks (hyperkalemic) and 26.6 +/- 1.7 weeks (non-hyperkalemic). The mean rate of increases in serum potassium levels was 0.32 +/- 0.29 mEq/L/hr (hyperkalemic) and 0.13 +/- 0.12 mEq/L/hr (non-hyperkalemic). The incidence of severe intraventricular hemorrhage (IVH) was 19% (18/93) (hyperkalemic) and 4.4% (3/68) (non-hyperkalemic). The incidence of cardiac arrhythmia was 12% (11/93) (hyperkalemic) and 0% (non-hyperkalemic) respectively. Neonates with fewer weeks of gestation at birth and faster increases in serum potassium levels were associated with a more prominent tendency toward hyperkalemia. Hyperkalemia markedly increases the risk of severe IVH and arrhythmia for ELBW infants. A higher glucose infusion rate should be maintained to prevent hypoglycemia following insulin treatment.

Continuous insulin intravenous infusion therapy for VLBW infants.

Very low birth weight (VLBW) infants less than 1,000 g often experience hyperkalemia and hyperglycemia during the initial hospital course. Hyperkalemia has been noted in 44% to 50% of infants less than 800 g birth weight or less than 28 to 29 weeks' gestation. Hyperglycemia occurs 18 times more frequently in infants less than 1,000 g than in those weighing more than 2,000 g. Insulin has been used for VLBW infants less than 1,000 g to manage hyperkalemia, control hyperglycemia, and optimize parenteral nutrition. A protocol for using exogenous insulin therapy for VLBW infants is described.

Glucose and insulin infusion versus kayexalate for the early treatment of non-oliguric hyperkalemia in very-low-birth-weight infants.

Forty very low birth weight (VLBW) infants with non-oliguric hyperkalemia in the first few days after birth were enrolled in this study. They were randomly divided into 2 groups, regular insulin (RI) infusion group and kayexalate resin enema group. Therapy was administered when serum potassium level was greater than 6 mEq/L. None of these infants received blood transfusion during this study course. In RI group (n = 20), the ratio of infusion glucose to regular insulin was 10-15 gm glucose to 1 unit RI, and the glucose infusion rate was maintained at least 6 mg/Kg/min. In Kayexalate group (n = 20), the dose of Kayexalate was 1 gm/Kg body weight given rectally every four hours. All treatment discontinued after the serum potassium level returned to normal for 6 hours. The mean gestational ages were 27.4 +/- 1.8 weeks in RI group and 28.4 +/- 2.4 weeks in Kayexalate group, respectively. Mean birth weights were 935 +/- 259 gm (RI) and 1065 +/- 214 gm (Kayexalate). The ages at onset of hyperkalemia were 24.6 +/- 8.2 (RI) and 22.2 +/- 8.1 (Kayexalate) hours after birth. The mean urine outputs during the 8-hour interval prior to development of hyperkalemia were 5.4 +/- 1.3 (RI) and 5.5 +/- 0.9 (Kayexalate) ml/kg/min. The durations of hyperkalemia were 26.4 +/- 14.9 (RI) and 38.6 +/- 13.3 (Kayexalate) hours. The peak serum potassium levels during therapy were 7.3 +/- 0.9 and 7.4 +/- 0.6 mEq/L. The incidences of grade II and above intraventricular hemorrhage (IVH) were 15% (3/20) and 50% (10/20). The incidences of cardiac dysrhythmia were 5% (1/20) and 10% (2/20). Significantly shorter duration of non-oliguric hyperkalemia and lower incidence of IVH were noted in RI group, but there were no differences in the peak potassium level or the incidence of cardiac dysrhythmia between these two groups. We conclude that to use early continuous regular insulin infusion therapy for the treatment of non-oliguric hyperkalemia in VLBW infants is more effective than kayexalate in decreasing the duration of hyperkalemia and reducing the incidence of intraventricular hemorrhage.

[2 newborn infants with severe arrhythmia caused by hyperkalemia]. / Twee pasgeborenen met een ernstige hartritmestoornis veroorzaakt door hyperkaliëmie.

Two newborn infants with ventricular arrhythmias secondary to hyperkalaemia are presented. One child also showed a decreased serum calcium concentration. There is scanty literature concerning the often life threatening cardiac arrhythmias due to hyperkalaemia in the newborn infants. Treatment of the cardiac arrhythmias require intravenous calcium gluconat and sodium bicarbonate infusion beside lowering the serum potassium level in the usual way.