Ashutosh Lal

Heterogeneity of Hemoglobin H Disease in Childhood

BACKGROUND Early diagnosis during newborn screening or infancy has enabled the observation of the natural history of hemoglobin H disease, a subtype of α-thalassemia. METHODS We analyzed longitudinal clinical data for patients with hemoglobin H disease arising from the deletion of three of four α-globin genes (HbH) and from hemoglobin H Constant Spring (HCS), caused by the deletion of two α-globin genes and the Constant Spring mutation. RESULTS We identified 86 patients with hemoglobin H disease (48 through newborn screening). Of these patients, 60 (70%) had HbH, 23 (27%) had HCS, and 3 (3%) had other, nondeletional forms of hemoglobin H disease. The parental ethnic background was Asian in 81% of patients, Hispanic in 5%, and African American in 3%, whereas mixed ancestry was observed in 10% of patients. Among the patients with deletional hemoglobin H disease, 15% had one or both parents with African-American ancestry. Growth was normal in patients with HbH during the first decade, but growth deficits began during infancy in those with HCS. Anemia was more severe in patients with HCS at all ages (P<0.001). Acute worsening of anemia with infections requiring urgent blood transfusion was observed in patients with HCS but not in those with HbH. The probability of receiving at least one transfusion by the age of 20 years was 3% for patients with HbH and 80% for those with HCS (P<0.001). Among patients with HCS, transfusions occurred in 13% of infants and 50% of children under the age of 6 years; splenectomy was associated with a significant improvement in hemoglobin levels (P=0.01) and a reduction in the number of transfusions. CONCLUSIONS HCS should be recognized as a distinct thalassemia syndrome with a high risk of life-threatening anemia during febrile illnesses. HbH was not associated with an increased rate of severe anemia with infections and was managed without blood transfusions. Many patients with these disorders had mixed ethnic backgrounds, which highlights the need for extended newborn screening in populations that are traditionally considered to be at low risk for hemoglobin H disease.

Bone mineral density in children with sickle cell anemia.

We evaluated bone mineral density (BMD) and risk factors for poor bone mineralization in children with sickle cell anemia (SCA).

Combined chelation therapy with deferasirox and deferoxamine in thalassemia.

Iron overload is the primary cause of mortality and morbidity in thalassemia major despite advances in chelation therapy. We performed a pilot clinical trial to evaluate the safety and efficacy of combined therapy with deferasirox (DFX, 20-30 mg/kg daily) and deferoxamine (DFO, 35-50mg/kg on 3-7 days/week) in 22 patients with persistent iron overload or organ damage. In the 18 subjects completing 12 months of therapy, median liver iron concentration decreased by 31% from 17.4 mg/g (range 3.9-38.2mg/g) to 12.0mg/g (range 0.96-26.7 mg/g, p<0.001). Median ferritin decreased by 24% from 2465 ng/mL (range 1110-10,700 ng/mL) to 1875 ng/mL (range 421-5800 ng/mL, p=0.002). All 6 subjects with elevated myocardial iron showed improvement in MRI T2* (p=0.031). The mean±S.E. plasma non-transferrin-bound iron (NTBI) declined from 3.10±0.25μM to 2.15±0.29μM (p=0.028). The administration of DFX during infusion of DFO further lowered NTBI (-0.28±0.08 μM, p=0.004) and labile plasma iron (LPI, -0.03±0.01 μM, p=0.006). The simultaneous administration of DFO and DFX rapidly reduced systemic and myocardial iron, and provided an excellent control of the toxic labile plasma iron species without an increase in toxicity.

Clinical assay of four thiol amino acid redox couples by LC–MS/MS: Utility in thalassemia☆

The total concentrations of four sulfur amino acid (SAA) metabolite redox couples (reduced and oxidized forms of homocysteine, cysteine, glutathione, and cysteinylglycine) in human blood are assayed with a simple and sensitive method by liquid chromatography-electrospray positive ionization-tandem mass spectrometry. To prevent ex vivo thiol oxidation, iodoacetamide (IAM) is used immediately following the blood draw. To selectively enrich for S-carboxyamidomethylated SAA, and other cationic amino acids metabolites, proprietary strong cation-exchange solid phase extraction tips are used. Analytes are further derivatized with isopropylchloroformate (IPCF) to esterify the amino and the carboxylic groups. Double derivatization with IAM and IPCF improves the reverse phase liquid chromatography separation of SAA metabolites. The use of detection mode of multiple-reaction monitoring (MRM) allows sensitive and specific simultaneous detection of SAA. The internal standards used to account for the matrix effects of human plasma and erythrocytes were plant glutathione analogue, homoglutathione, and stable isotopes of cystine and homocystine. The method was validated for its linearity, accuracy, and precision. Excellent linearity of detection (r(2)>0.98) was observed over relevant ranges for plasma and erythrocyte samples, and the limits of detection were established to be between 5 and 20nM. Relative standard deviations were <9% for within-day variations and <15% for between-day variations. The method was used to assess thiol redox states in plasma and erythrocytes isolated from healthy subjects and thalassemia patients.

Association of chromosome damage detected as micronuclei with hematological diseases and micronutrient status

Epidemiological studies reveal strong association between micronutrient deficiencies and development of cancer. Since chromosome breaks and abnormal chromosome segregation, identified as micronuclei (MN), are central to malignant transformation, the influence of micronutrient status upon MN frequency has been the subject of intense research. Motivating this effort is the idea that marginal micronutrient deficiencies lead to allocation of scarce cellular resources towards immediate survival at the expense of maintaining genomic integrity, placing the individual at greater risk for degenerative diseases and cancer in old age. The challenge in identifying an association between individual micronutrients and MN frequency stems from the complexity of human diet, simultaneous presence of multiple micronutrient deficiencies, variable genetic susceptibility and methodological difficulties. A unique model for studying MN in humans is provided by a group of haematological diseases, the chronic haemolytic anaemias associated with high reticulocyte count and absence of splenic function. These disorders may prove valuable for assessing the influence of micronutrient status once the effect of abnormal erythropoiesis on MN formation is adequately understood. Eventually, large population-based studies that can account for the baseline variability in MN frequency, lifestyle and genetic factors may be needed to uncover the DNA-damaging effect of poor diet. Understanding the link between micronutrient status and MN frequency will contribute towards determining optimal micronutrient intake to preserve long-term health.

Treatment of vitamin D deficiency in transfusion-dependent thalassemia†

The survival of patients with thalassemia major has progressively improved with advances in therapy; however, osteoporosis remains a frequent, unresolved issue [1]. Adequate circulating levels of vitamin D are essential for optimal skeletal health and reducing fracture risk [2]. Vitamin D insufficiency is reported in the majority of patients with thalassemia in the USA [3] and elsewhere [4–10], despite routine prescription of 400–1,000 IU vitamin D per day. In this study, assessment of serum 25-hydroxy vitamin D (25-OH D) levels in 96 patients with thalassemia revealed that 70 (73%) were either deficient ( 80 ng/ml was observed over the course of the observation period. There were 18 individuals who attained a 25-OH D level >30 ng/ml at the end of their supplementation period and continued daily supplementation of 400–1,000 IU vitamin D thereafter. When retested at an average of 8 months (1–21 months) later, the serum 25-OH D had dropped significantly and collectively for all but two of them, from a mean of 34.4 ± 3.7 to 26.9 ± 6.8 ng/ml (P < 0.001). The rate of decline was on an average 1.5 ng/ml per month. Hence, patients who had inadequate vitamin D status on screening were likely to require ongoing high-dose supplementation. In contrast, the

Lipoic acid and acetyl-carnitine reverse iron-induced oxidative stress in human fibroblasts.

Abstract Iron overload occurs frequently in thalassemia and other disorders that require regular blood transfusions. Excess iron is toxic owing to the generation of free radicals that lead to oxidation of biomolecules and tissue damage. In order to identify compounds that reduce oxidative injury from iron, we evaluated α-lipoic acid (LA), a multifunctional antioxidant, in iron-overloaded primary human fibroblasts (IMR-90). Oxidant stress was measured using dichlorodihydrofluorescein diacetate that is converted to the fluorescent dichlorofluorescein (DCF) upon oxidation. Exposure to ferric ammonium citrate (FAC) increased the iron-content of IMR-90 cells and caused a rise in oxidant appearance. The addition of LA improved the cellular redox status and attenuated the iron-mediated rise in oxidants in a dose-dependent manner. The R- and RS-enantiomers of LA demonstrated similar antioxidant activity. N-tert-butyl hydroxylamine (NtBHA) treated cells also exhibited a decrease in DCF fluorescence, but at a much higher concentration compared with LA. The combination acetyl-L-carnitine (ALCAR) and LA exhibited superior antioxidant effect at all dose levels. We conclude that LA is highly effective in reversing oxidative stress arising from iron overload and that its antioxidant efficacy is further enhanced in combination with ALCAR.

A nutrient-dense, high-fiber, fruit-based supplement bar increases HDL cholesterol, particularly large HDL, lowers homocysteine, and raises glutathione in a 2-wk trial

Dietary intake modulates disease risk, but little is known how components within food mixtures affect pathophysiology. A low‐calorie, high‐fiber, fruit‐based nutrient‐dense bar of defined composition (e.g., vitamins and minerals, fruit polyphenolics, β‐glucan, docosahexaenoic acid) appropriate for deconstruction and mechanistic studies is described and evaluated in a pilot trial. The bar was developed in collaboration with the U.S. Department of Agriculture. Changes in cardiovascular disease and diabetes risk biomarkers were measured after 2 wk twice‐daily consumption of the bar, and compared against baseline controls in 25 healthy adults. Plasma HDL‐cholesterol (HDL‐c) increased 6.2% (P=0.001), due primarily to a 28% increase in large HDL (HDL‐L; P<0.0001). Total plasma homocysteine (Hcy) decreased 19% (P=0.017), and glutathione (GSH) increased 20% (P=0.011). The changes in HDL and Hcy are in the direction associated with decreased risk of cardiovascular disease and cognitive decline; increased GSH reflects improved antioxidant defense. Changes in biomarkers linked to insulin resistance and inflammation were not observed. A defined food‐based supplement can, within 2 wk, positively impact metabolic biomarkers linked to disease risk. These results lay the groundwork for mechanistic/deconstruction experiments to identify critical bar components and putative synergistic combinations responsible for observed effects.—Mietus‐Snyder, M. L., Shigenaga, M. K., Suh, J. H., Shenvi, S. V., Lal, A., McHugh, T., Olson, D., Lilienstein, J., Krauss, R. M., Gildengoren, G., McCann, J. C., Ames, B. N. A nutrient‐dense, high‐fiber, fruit‐based supplement bar increases HDL cholesterol, particularly large HDL, lowers homocysteine, and raises glutathione in a 2‐wk trial. FASEB J. 26, 3515–3527 (2012). www.fasebj.org

Zinc Status Affects Glucose Homeostasis and Insulin Secretion in Patients with Thalassemia

Up to 20% of adult patients with Thalassemia major (Thal) live with diabetes, while 30% may be zinc deficient. The objective of this study was to explore the relationship between zinc status, impaired glucose tolerance and insulin sensitivity in Thal patients. Charts from thirty subjects (16 male, 27.8 ± 9.1 years) with Thal were reviewed. Patients with low serum zinc had significantly lower fasting insulin, insulinogenic and oral disposition indexes (all p < 0.05) and elevated glucose response curve, following a standard 75 g oral load of glucose compared to those with normal serum zinc after controlling for baseline (group × time interaction p = 0.048). Longitudinal data in five patients with a decline in serum zinc over a two year follow up period (−19.0 ± 9.6 μg/dL), showed consistent increases in fasting glucose (3.6 ± 3.2 mg/dL) and insulin to glucose ratios at 120 min post glucose dose (p = 0.05). Taken together, these data suggest that the frequently present zinc deficiency in Thal patients is associated with decreased insulin secretion and reduced glucose disposal. Future zinc trials will require modeling of oral glucose tolerance test data and not simply measurement of static indices in order to understand the complexities of pancreatic function in the Thal patient.

Measuring Chromosome Breaks in Patients with Thalassemia

Abstract: Iron‐mediated oxidative stress plays an important role in the pathophysiology of thalassemia. Oxidative stress can cause lesions in DNA, including double‐strand breaks. DNA damage, which is a cause of cancer (although not the only one), is recognized as deleterious. Unlike cancer, DNA damage can be assayed easily and relatively inexpensively in humans. In this study, a sensitive micronucleus assay was used to measure the frequency of chromosomal breaks in patients with α‐ and β‐thalassemia. The micronucleus test is based on the observation that a secondary nucleus (micronucleus) is formed around a chromosomal fragment, outside the main nucleus of a dividing cell. Micronuclei are readily apparent in red blood cells (RBCs), which otherwise lack DNA. We combined an immunomagnetic separation technique with single‐laser flow cytometry to isolate and analyze reticulocytes in peripheral blood for the presence of micronuclei before these cells are removed by the spleen. Blood samples were obtained from patients with thalassemia and healthy volunteers. After immunomagnetic enrichment of CD71‐positive reticulocytes, the cells were stained for micronuclei using the DNA dye 7‐aminoactinomycin D (7‐AAD) and evaluated by flow cytometry. Our findings indicate that higher levels of micronuclei frequencies are present in thalassemic RBCs.