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Patient comprehension of emergency department (ED) discharge instructions is important for ensuring that patients understand their diagnosis, recommendations for treatment, appropriate follow-up, and reasons to return. However, many patients may not fully understand their instructions. Furthermore, some patients may state they understand their instructions even when they do not. We surveyed 75 patients on their perception of their understanding of their ED discharge instructions, and asked them specific questions about the instructions. We also performed a chart review, and examined patients' answers for correlation with the written instructions and medical chart. We then performed a statistical analysis evaluating which patients claimed understanding but who were found to have poor understanding on chart review. Overall, there was no significant correlation between patient self-reported understanding and physician evaluation of their understanding (ρ = 0.221, p = 0.08). However, among female patients and patients with less than 4 years of college, there was significant positive correlation between self-report and physician evaluation of comprehension (ρ = 0.326, p = 0.04 and ρ = 0.344, p = 0.04, respectively), whereas there was no correlation for male patients and those with more than 16 years of education (ρ = 0.008, p = 0.9, ρ = -0.041, p = 0.84, respectively). Patients' perception of their understanding may not be accurate, especially among men, and those with greater than college education. Identifying which patients say they understand their discharge instructions, but may actually have poor comprehension could help focus future interventions on improving comprehension.

Objectives Point-of-care ultrasound (POCUS) may facilitate the diagnosis and management of children for various conditions. Integration of POCUS into clinical care requires hands-on training; however, providers may be reluctant to perform educational ultrasounds to improve their skills, as it is a procedure without direct clinical benefit to the patient and due to concerns that it may lower a family's overall satisfaction. We sought to evaluate whether the use of POCUS changed overall patient/caregiver satisfaction in a pediatric emergency department (ED) visit. Methods We performed a prospective cohort study of children presenting to a single children's hospital ED. We evaluated overall satisfaction for three groups: 1) children undergoing POCUS for educational purposes, 2) children undergoing POCUS for a specific diagnostic indication, and 3) controls who did not receive POCUS. Overall satisfaction was measured using a 100-point visual analog scale (VAS). We performed a noninferiority test between patients receiving POCUS and controls using a delta of 10 points to determine significance. We also examined satisfaction among patients receiving diagnostic ultrasound and sought to determine patient/caregiver satisfaction with specific elements of the POCUS experience. Results We surveyed 159 patients who presented between April 2016 and August 2016 (53 in each group). The three groups did not differ with respect to patient age, sex, or Emergency Severity Index level. The median VAS scores for both the educational and the diagnostic POCUS groups (94 and 94, respectively) were not inferior to the VAS score for the control group (91) with a median (95% confidence interval [CI]) difference of 3.0 (-2.2 to 8.2) for educational and control groups and 3.0 (-1.6 to 7.6) for diagnostic and control groups. No patients/caregivers in the educational POCUS group would refuse an educational ultrasound during a future medical encounter. Conclusion Educational POCUS does not decrease patient/caregiver satisfaction among children presenting to the ED.

Background Point-of-care ultrasound (POCUS) is increasingly being used to differentiate cellulitis from abscess in the pediatric emergency department (ED). POCUS has also been shown to decrease length of stay (LOS) for other applications among adult patients. Objective We sought to determine if ED LOS differed for children presenting with skin and soft tissue infections who received POCUS versus radiology-performed ultrasound. Methods We performed a retrospective cohort study of children presenting to an urban pediatric ED between January 2011 and June 2013 with a diagnosis of cellulitis or abscess who underwent soft tissue ultrasound. Patients who received a surgical consult, had significant medical comorbidities, or had a lesion located on the face, hands, feet, or groin were excluded. We compared ED LOS among children who received radiology-performed ultrasound to children who received POCUS, adjusting for relevant clinical variables. Results Among 3094 children with a diagnosis of cellulitis or abscess, we identified 202 who underwent a POCUS and 118 who underwent radiology-performed ultrasound. The POCUS group had a shorter median LOS than the radiology-performed ultrasound group (adjusted median difference –73 min; 95% confidence interval –93.6 to –52.4 min). In the subset of patients discharged from the ED, this difference was more pronounced (adjusted median difference –89 min; 95% confidence interval –109.9 to –68.1 min). Conclusion Among children presenting to a pediatric ED with superficial skin and soft tissue infections, children receiving POCUS experienced shorter LOS compared to children receiving radiology-performed ultrasound.

Rising health care costs demands justifying prolonged hospitalization for syncope, yet predictors of shorter length of stay (LOS) have not been identified.The objective of this study is to identify independent predictors of LOS for syncope patients presenting to the emergency department (ED).We performed an analysis of a prospectively collected cohort of ED syncope patients. We examined risk factors from the patient's ED presentation and workup and used Spearman rank correlation to evaluate the relationship between these risk factors and LOS in a logistic regression model for prediction of hospitalization less than 1 day.Of 568 total syncope patients, 350 (61.6%) were admitted and comprise the study cohort. Mean age was 57 years (SD, ±25 years); 39.6% were male. Length of hospitalization was less than or equal to 1 in 179 patients (51.1%). The number of risk factors was associated with LOS (P.001). Most hospitalized patients with less than 1 predictor were hospitalized for less than 1 day (152/272; 55.9%). In multivariate logistic regression analysis, clear vasovagal etiology was positively correlated with LOS less than 1 day (odds ratio [OR], 1.92), whereas dysrhythmia (OR, 0.3), coronary artery disease (OR, 0.33), abnormal vital signs (OR, 0.35), implantable defibrillator/pacemaker (OR, 0.29), anemia/gastrointestinal bleeding (OR, 0.34), and central nervous system abnormalities (OR, 0.09; P.05 for all) were negatively correlated with LOS less than 1 day. Area under the curve for model accuracy was moderate: 0.70 (95% confidence interval, 0.65-0.76).Patients with less than 1 predictor of prolonged stay, who present to the ED with syncope, are unlikely to require hospitalization of more than 1 day. A set of predictors may help define which patients are appropriate for 24-hour observation rather than full hospitalization.

Objectives The objective was to quantify the correlation between general numeracy and health literacy in an emergency department (ED) setting. Methods This was a prospective cross-sectional convenience sample study of adult patients in an urban, academic ED with 97,000 annual visits. General numeracy was evaluated using four validated questions and health literacy using three commonly used validated screening tools (Short Test of Functional Health Literacy in Adults [S-TOFHLA], Rapid Estimate of Adult Literacy in Medicine-Revised [REALM-R], and the Newest Vital Sign [NVS]). Scores were dichotomized for health literacy tests to limited (low or marginal) versus adequate health literacy, and the proportion of patients answering all numeracy questions correctly was calculated with the mean proportion of correct responses in these groups. The correlation between numeracy scores and scores on the health literacy screening tools was evaluated using Spearman's correlation. Results A total of 446 patients were enrolled. Performance on questions evaluating general numeracy was universally poor. Only 18 patients (4%) answered all numeracy questions correctly, 88 patients (20%) answered zero questions correctly, and overall the median number of correct answers was one (interquartile range [IQR] = 1 to 2). Among patients with limited health literacy (LHL) by any of the three screening tools used, the mean number of correct numeracy answers was approximately half that of patients with adequate health literacy. However, even among those with adequate health literacy, the average number of correct answers to numeracy questions ranged from 1.6 to 2.4 depending on the screening test used. When dichotomized into those who answered ≤50% versus >50% of numeracy questions correctly, there was a significant difference between those with LHL and those who scored ≤50% on numeracy. Health literacy screening results were correlated with general numeracy in the low to moderate range: S-TOFHLA rs = 0.428 (p

Objectives: Improper placement of the tracheal tube during intubation can lead to dangerous complications, and bedside ultrasound has been proposed as a method of quickly and accurately identifying tube placement. Recent studies in adults have found it to be accurate, but its applicability in pediatric patients is unclear. This systematic review aims to describe the current available data on the accuracy and feasibility of bedside ultrasound for tracheal tube placement in children. Data Sources: OVID MEDLINE and EMBASE. Study Selection: Available articles on bedside neck or lung/diaphragm ultrasound for confirmation of tracheal tube placement in children through December 2015. Data Extraction: Two reviewers screened studies for eligibility and abstracted data independently. The quality of selected articles was evaluated using Quality Assessment of Diagnostic Accuracy Studies statement. Data Synthesis: A total of nine articles were identified: one study using neck ultrasound, two using lung/diaphragmatic ultrasound, one with both, and five studies looking at direct visualization of the tracheal tube tip met our inclusion criteria. There were 81 intubations evaluated using neck ultrasound, 214 intubations evaluated using diaphragmatic or pleural sliding, and 165 intubations evaluated for feasibility of bedside ultrasound in visualizing tracheal tube tip placement. The sensitivities of transtracheal ultrasound for intubation were overall high ranging from 0.92 to 1.00 with excellent specificities at 1.00. For lung ultrasound, the sensitivities for tracheal placement versus esophageal placement were high at 1.00, but only one study reported esophageal intubations and had a specificity of 1.00. When assessing the appropriate tracheal tube depth for tracheal intubations using lung ultrasound, the sensitivities ranged from 0.91 to 1.00 with specificities ranging from 0.5 to 1.0. Regarding feasibility of direct visualization of tracheal tube tip, visualization ranged from 83% to 100%. Conclusion: Bedside ultrasound has been described to be feasible in determining tracheal tube placement in several small single center studies and could be a useful adjunct tool in confirming tracheal tube placement in critically ill pediatric patients, but further studies are needed to assess its accuracy in a randomized multicenter setting.

Purpose Global data on human papillomavirus (HPV) serological and deoxyribonucleic acid (DNA) prevalence are essential to optimize HPV prophylactic vaccination strategies. Methods We conducted a global review of age-specific HPV antibody and studies with both antibody and DNA prevalence for HPV-16, −18, −6, and −11. Results One hundred seventeen studies were included; participants' ages ranged from several hours to >90 years. HPV-16 seroprevalence was generally higher in Africa, Central and South America, and North America, more prevalent among women than among men, and peaked around ages 25–40 years. HPV-18 seroprevalence was generally lower than HPV-16 with a later age peak. Data were limited for HPV-6 and −11, both of which peaked at ages similar to HPV-18. Among 9–26-year-old females, HPV-16 seroprevalence ranged from 0%–31% in North America, 21%–30% in Africa, 0%–23% in Asia/Australia, 0%–33% in Europe, and 13%–43% in Central and South America. HPV-16/-18 DNA prevalence peaked 10–15 years before corresponding HPV-16/-18 antibody prevalence. Conclusions Females within the HPV vaccine-eligible age-group (9–26 years) had a range of dual HPV-16 DNA and serology negativity from 81%–87%, whereas 90%–98% were HPV-16 DNA negative. Serology and DNA data are lacking worldwide for females younger than age 15 years, the prime target group for vaccination.

Objectives The objective was to evaluate the diagnostic accuracy of five health literacy screening instruments in emergency department (ED) patients: the Rapid Evaluation of Adult Literacy in Medicine-Revised (REALM-R), the Newest Vital Sign (NVS), Single Item Literacy Screens (SILS), health numeracy, and physician gestalt. A secondary objective was to evaluate the feasibility of these instruments as measured by administration time, time on task, and interruptions during test administration. Methods This was a prospective observational cross-sectional study of a convenience sampling of adult patients presenting during March 2011 and February 2012 to one urban university-affiliated ED. Subjects were consenting non–critically ill, English-speaking patients over the age of 18 years without aphasia, dementia, mental retardation, or inability to communicate. The diagnostic test characteristics of the REALM-R, NVS, SILS, health numeracy, and physician gestalt were quantitatively assessed by using the short Test of Functional Health Literacy in Adults (S-TOFHLA). A score of 22 or less was the criterion standard for limited health literacy (LHL). Results A total of 435 participants were enrolled, with a mean (±SD) age of 45 (±15.7) years, and 18% had less than a high school education. As defined by an S-TOFHLA score of 22 or less, the prevalence of LHL was 23.9%. In contrast, the NVS, REALM-R, and physician gestalt identified 64.8, 48.5, and 35% of participants as LHL, respectively. A normal NVS screen was the most useful test to exclude LHL, with a negative likelihood ratio of 0.04 (95% confidence interval [CI] = 0.01 to 0.17). When abnormal, none of the screening instruments, including physician gestalt, significantly increased the posttest probability of LHL. The NVS and REALM-R require 3 and 5 minutes less time to administer than the S-TOFHLA. Administration of the REALM-R is associated with fewer test interruptions. Conclusions One-quarter of these ED patients had marginal or inadequate health literacy. Among the brief screening instruments evaluated, a normal NVS result accurately reduced the probability of LHL, although it will identify two-thirds of ED patients as high risk for LHL. None of the brief screening instruments significantly increases the probability of LHL when abnormal. Resumen Objetivos Evaluar la certeza diagnostica de cinco instrumentos de despistaje de educacion sanitaria en los pacientes del servicio de urgencias (SU): el Rapid Evaluation of Adult Literacy in Medicine-Revised (REALM-R), el Newest Vital Sign (NVS), el Single Item Literacy Screens (SILS), los calculos numericos relacionados con la salud y un medico gestaltico. Un objetivo secundario fue evaluar la viabilidad de estos instrumentos medida por el tiempo de administracion, el tiempo en la tarea y las interrupciones durante la administracion del test. Metodologia Estudio transversal de una muestra de conveniencia de pacientes adultos que acudieron durante marzo de 2011 y febrero de 2012 a un SU universitario y urbano. Los sujetos que consintieron fueron enfermos no criticos, de habla inglesa y mayores de 18 anos de edad sin afasia, demencia, retraso mental o incapacidad para comunicarse. Las caracteristicas de los tests diagnosticos REALM-R, NVS, SILS y calculo numerico relativo a la salud y del medico gestaltico se evaluaron cuantitativamente mediante la forma abreviada del Test of Functional Health Literacy in Adults (S-TOFHLA). Una puntuacion de 22 o menos fue el criterio estandar para considerar una educacion sanitaria limitada (ESL). Resultados Se incluyeron 435 participantes, con una media de edad de 45 anos (DE), y un 18% tenia una formacion inferior a la escuela secundaria. Como se definio por una puntuacion de S-TOFHLA de 22 o menos, la prevalencia de ESL fue un 23,9%. En comparacion, el NVS, el REALM-R, y el medico gestaltico identificaron un 64,8%, un 48,5%, y un 35% de los participantes como ESL, respectivamente. Un NVS normal fue el test mas util para excluir ESL, con una razon de probabilidad negativa de 0,04 (IC 95% = 0,01 a 0,17). Cuando fue anormal, ninguno de los instrumentos de despistaje, incluyendo el medico gestaltico, incremento significativamente la probabilidad post-test de ESL. El NVS y el REALM-R requieren 3 y 5 minutos menos de tiempo para administrase que el S-TOFHLA. La administracion del REALM-R se asocia con menos interrupciones del test. Conclusiones Una cuarta parte de los pacientes del SU tiene una educacion sanitaria inadecuada o marginal. Entre los instrumentos de despistaje breves evaluados, un resultado normal en el NVS redujo certeramente la probabilidad de una ESL, aunque identificara dos tercios de los pacientes del SU como de alto riesgo para una ESL. Ninguno de los instrumentos breves de despistaje incrementa significativamente la probabilidad de ESL cuando es anormal.

Compound heterozygotes of variant haemoglobins (Hbs) with HbC, with or without novel phenotypic changes, have provided insight into the molecular basis of the interacting haemoglobins and information concerning the role of specific residues in the crystallization of oxy HbC. A high phosphate buffer system has proved useful for studying the effects of variant haemoglobins (naturally co-existing with HbC in the red cell) on the oxy HbC crystallization process and has led us to conclude that beta87 and beta73 are contact sites of the oxy HbC crystal. We now present investigations from two HbC compound heterozygotes which exhibit opposing effects upon HbC crystallization: HbC/Hb N-Baltimore (beta95 Lys--Glu) and HbC/Hb Riyadh (beta120 Lys--Asn). The latter inhibits the in vitro crystallization of HbC, explaining the lack of erythrocyte abnormalities (with the exception of microcytosis) in the doubly heterozygous infant. In contrast, Hb N-Baltimore accelerates the crystallization of HbC, contributing to multiple abnormalities in red cell morphology, albeit in the absence of morbidity. We conclude that (1) beta120 and beta95 are additional contact sites in the crystal, and (2) the HbC/Hb Riyadh haemoglobinopathy demonstrates that crystallization may not be required for the generation of the observed microcytosis and increased red cell density in HbC-containing red cells.

Velo-cardio-facial syndrome (VCFS) is a congenital anomaly characterized by multiple dysmorphisms, cleft palate, cardiac anomalies, and learning disabilities, that results from a microdeletion of chromosome 22q11. An increased prevalence of psychiatric illness has been observed, with both schizophrenia and bipolar disorder commonly being diagnosed. For these reasons, the VCFS region is an interesting candidate region for bipolar disorder. We examined this region in 17 bipolar families from three populations: 13 families from the general North American population (University of California, San Diego/University of British Columbia, UCSD/UBC), three larger families from New York, and a portion of Old Order Amish pedigree 110. Three microsatellite markers spanning 13 cM around the VCFS region were genotyped in all the families. A maximum lod score of 2.51 was obtained in the UCSD/UBC families under a dominant model at D22S303. In the combined family set, maximum lod scores of 1.68 and 1.28 were obtained at this marker under dominant and recessive models, respectively. Four additional markers were subsequently typed in selected positive families, and yielded positive lods at 6 of 7 markers spanning 18 cM in this region. Nonparametric, multipoint analyses using the affected pedigree member (APM) method also yielded suggestive evidence for linkage in both the UCSD/UBC family set (P = 0.0024) and in the combined families (P = 0.017). Affected sibpair analyses were similarly positive in the UCSD/UBC families (P = 0.017), and in the combined families (P = 0.004). These results are suggestive of a possible locus for bipolar disorder near the VCFS region on chromosome 22.

We report here that compound heterozygosity for hemoglobin Korle-Bu (HbKB) and HbC (beta 6 Glu-->Lys) is associated with moderate chronic hemolytic anemia with microcytosis. To understand the pathogenesis of this syndrome, we have studied the effect of Hb Korle-Bu (KB = beta 73 Asp-->Asn) on the crystallization of HbC. We have previously established that fetal Hb (HbF) inhibits the crystallization of HbC. In contrast, HbS accelerates crystallization affecting the pathogenesis of SC disease. We now report on in vitro crystallization of mixtures of HbKB, HbC, and various amounts of HbF and the native hemolysate of a child who is a compound heterozygote for HbKB and HbC. At 6 months of age, the propositus' hemolysate contained 55% HbKB, 39% HbC, and 6% HbF. Crystal formed within 2 minutes compared with 30 minutes for the mixture of 40% HbC:60% HbS and with 180 minutes for 40% HbC:60% HbA. The morphology of the crystals formed was cubic, in contrast with the tetragonal crystals observed in CC and SC disease. Early crystals did not exhibit “sharp edges” until 45 minutes. Purified HbKB formed aggregates but not crystals after 24 hours. Isopycnic gradients showed that the KB/C compound heterozygotes have red blood cell (RBC) densities intermediate between the AC and CC phenotype and similar to SC disease. The surface residue beta 73, known to participate in areas of interaction of the deoxy HbS polymer, can now be assigned to areas of contact in HbC containing crystals. The hemolysis observed in the HbKB/C compound heterozygote is likely to be secondary to the acceleration of Hb crystallization. The microcytosis and increased RBC density is clearly the consequence of the presence of HbC, but the basis of the increased RBC pathology compared with AC trait, despite the low proportion of HbC (35% to 40%), remains to be elucidated.

We report here that compound heterozygosity for hemoglobin Korle-Bu (HbKB) and HbC (beta 6 Glu-->Lys) is associated with moderate chronic hemolytic anemia with microcytosis. To understand the pathogenesis of this syndrome, we have studied the effect of Hb Korle-Bu (KB = beta 73 Asp-->Asn) on the crystallization of HbC. We have previously established that fetal Hb (HbF) inhibits the crystallization of HbC. In contrast, HbS accelerates crystallization affecting the pathogenesis of SC disease. We now report on in vitro crystallization of mixtures of HbKB, HbC, and various amounts of HbF and the native hemolysate of a child who is a compound heterozygote for HbKB and HbC. At 6 months of age, the propositus' hemolysate contained 55% HbKB, 39% HbC, and 6% HbF. Crystal formed within 2 minutes compared with 30 minutes for the mixture of 40% HbC:60% HbS and with 180 minutes for 40% HbC:60% HbA. The morphology of the crystals formed was cubic, in contrast with the tetragonal crystals observed in CC and SC disease. Early crystals did not exhibit “sharp edges” until 45 minutes. Purified HbKB formed aggregates but not crystals after 24 hours. Isopycnic gradients showed that the KB/C compound heterozygotes have red blood cell (RBC) densities intermediate between the AC and CC phenotype and similar to SC disease. The surface residue beta 73, known to participate in areas of interaction of the deoxy HbS polymer, can now be assigned to areas of contact in HbC containing crystals. The hemolysis observed in the HbKB/C compound heterozygote is likely to be secondary to the acceleration of Hb crystallization. The microcytosis and increased RBC density is clearly the consequence of the presence of HbC, but the basis of the increased RBC pathology compared with AC trait, despite the low proportion of HbC (35% to 40%), remains to be elucidated.

The structural basis of the crystallizing tendencies of oxyHbC (beta 6Glu-->Lys), that produces haemolytic anaemia in homozygotes, is unknown. Using a fluorescent organic phosphate analogue (8-hydroxy-1,3,6-pyrenetrisulphonate), and conventional oxygen equilibrium studies, data suggest that the binding of inositolhexaphosphate (IHP) to oxyHbC differs from HbA, indicating perturbations of the oxyHbC central cavity, which was predicted from our earlier spectroscopic findings. To define the relationship between this conformational change in oxyHbC and its tendency to crystallize, the effect of four central cavity ligands on the crystallization rate was studied: a peptide containing 11 residues from the N-terminal portion of band 3, the full cytoplasmic domain of band 3, 2,3-diphosphoglycerate and IHP. OxyHbC crystallization was accelerated by all these central cavity ligands and not by the appropriate controls. These central cavity changes become an excellent candidate for the dramatic increase in the crystallization rate of oxyHbC.

Hb Montefiore was found, in the heterozygous state, in a Puerto Rican female who had a slightly elevated total Hb level. Structural analysis revealed that Asp-α126 was replaced by Tyr. Hb Montefiore migrates close to HbF (at pH 8.6) and accounts for 20.3% of the hemolysate. Oxygen binding of red blood cells revealed a 40% decrease in the P50 (pH 7.4) and a low n value of 1.6 (normal: 2.6). Depletion of red blood cell 2,3-DPG did not change the results. Stripped Hb Montefiore at pH 7.2 showed an 8-fold reduction in P50 (0.6 versus 4.6 mm Hg) and very low cooperativity (n = 1.2 versus 2.9 for the control). Heterotopic effectors, as 2,3-diphosphoglycerate and inositol hexaphosphate had a normal effect and in addition, they increased cooperativity. The chloride ion effect and the Bohr effect were moderately reduced. A bezafibrate derivative (L345), known to bind α126, increases the P50 of HbA by 9-fold, but only by 1.5-fold that of Hb Montefiore. Combining these functional studies with intrinsic fluorescence and Resonance Raman spectroscopy, we interpret the very low n value and the high oxygen affinity for Hb Montefiore as a result of both a destabilized T state that switches to R upon ligand binding and a deoxy T state that binds ligands with higher affinity than that of deoxy HbA. Hb Montefiore still binds ligands cooperatively, but the difference in ligand binding properties of the two quaternary states has been drastically reduced.

Whole blood and hemolysates from seven normal and three erythropoietic protoporphyria patients were compared in terms of their hemoglobin function. The oxygen affinity (P50) of the erythropoietic protoporphyria hemolysates compared to normals (13.1 +/- 0.2 vs 17.5 +/- 0.3 mmHg; P < 0.001) and erythropoietic protoporphyria erythrocytes compared to normals (23.4 +/- 0.6 vs 27.1 +/- 0.5 mmHg; P < 0.001) were increased while oxygen-binding cooperativity (n-value of the Hill equation) were similar. We conclude that hemoglobin function in erythropoietic protoporphyria patients is altered, but without pathophysiologic consequences. Because hemoglobin in which protoporphyrin IX substitutes for heme has a low oxygen affinity, our findings of a higher than normal affinity in erythropoietic protoporphyria red cells and hemolysates may indirectly support the findings by others that protoporphyrin IX binds to hemoglobin at non-heme-binding sites. In addition, based on the effect of other abnormal hemoglobins, this shift in P50 will decrease any tendency for anemia in erythropoietic protoporphyria patients.

In erythropoietic protoporphyria and lead poisoning, free protoporphyrin (PPIX) and zinc protoporphyrin (ZPP), respectively, accumulate in erythrocytes. That PPIX and ZPP bind to human hemoglobin A (Hb4) is established, but the site of binding is still a matter of controversy. We investigated the interaction of ZPP with intact, tetrameric oxy Hb4, using batch microcalorimetry, front-face fluorometry, absorption difference spectroscopy, oxygen equilibrium studies, and isoelectric focusing (IEF). In the presence of oxy Hb4 (pH 7.35, 0.05 M phosphate), the fluorescence emission maximum (excitation at 420 nm) of ZPP immediately shifts from 587 nm (ZPP alone) to 594 nm, as expected when binding to protein. The fluorescence intensity increases with time and is correlated with the ZPP:Hb4 mole ratio. A slow, time-dependent reaction is also observed with microcalorimetry: the rate of heat of reaction exhibits both a fast and a slow component. The heats of reaction range from -2.1 to -14.8 mcal depending upon the ZPP:Hb4 ratio of 4:1 (0.4 mM:0.1 mM) to 38:1 (3.8 mM:0.1 mM), respectively, and are typical of weak, noncovalent protein-ligand interactions. The optical difference spectra are a function of the ZPP:Hb4 molar ratio and also exhibit a slow increase in intensity over time. No time-dependent optical difference spectra are observed with ZPP or with Hb4 alone. The oxygen affinity of Hb4 in the presence of ZPP decreases with increasing mole ratio. During IEF, all ZPP separates from Hb4, consistent with a weak, noncovalent interaction at a non-heme pocket site. We conclude that ZPP binds to intact, tetrameric hemoglobin at non-heme pocket sites in a nonspecific, weak, noncovalent interaction.

We have reported that circulating CC erythrocytes containing HbO2 C crystals exhibit little or no Hb F suggesting that Hb F may inhibit the crystallization of Hb C. We report now that Hb F inhibits in vitro crystallization of HbO2 and HbCO C when compared to the effect of Hb A in a wide range of mixture proportions. For example, while HbCO C solutions form tetragonal C crystals within 25 min, no crystals form within 2 h with 30% Hb F, whereas 550 crystals/mm3 form with 30% Hb A. Furthermore, an increase in the percent of Hb A is correlated with a greater number of orthorhombic crystal formation rather than the tetragonal morphology observed with 100% Hb C. We also report that Hb A2 (containing delta chains that exhibit 10 sequence differences with beta chains) and Hb Lepore Boston-Washington (a fusion mutant of delta and beta chains that contains only six of these differences) both inhibit Hb C crystallization. By comparing the sequences of the three inhibitory hemoglobins, we conclude that position Gln-87 in the gamma chains is, at least partially, the cause of the inhibitory effect of Hb F on the crystallization of Hb C.

We previously reported that circulating hemoglobin (Hb) CC erythrocytes contain oxygenated HbC crystals with little or no HbF and that HbF inhibits in vitro crystallization of HbC. We now report that HbS accelerates in vitro crystallization of HbC. Crystals were formed in 1.8 mol/L potassium phosphate buffer, pH 7.4, at 30 degrees C and were counted in several time intervals with a hematocytometer. The hemoglobin composition of Millipore-isolated crystals and supernatant were also analyzed. Under the conditions selected, 100% HbS formed needle-shaped crystals only after two hours. Pure HbC does not form crystals within 15 minutes, whereas a ratio of 10% HbS:90% HbC forms 1,100 crystals/mm3, 20% HbS:80% HbC forms 370 crystals/mm3, and 30% HbS:70% HbC forms 5 crystals/mm3. Crystals formed in the presence of HbS are tetragonal, as are pure HbC crystals. As compared with 100% HbC, HbA or albumin mixed with HbC showed a decreased number of crystals as a result of dilution. Analysis of the Hb content of isolated crystals by citrate agar gel electrophoresis showed that HbS was rapidly incorporated into the crystal in the same ratio over time. These results demonstrate that HbS accelerates crystallization of HbC with respect to the rates of crystallization of any of these two Hbs separately, through a mechanism that involves cocrystallization. These results may be significant in understanding SC disease.