Maria del Pilar Aguinaga

Adverse Effects of a Clinically Relevant Dose of Hydroxyurea Used for the Treatment of Sickle Cell Disease on Male Fertility Endpoints

Two experiments were conducted to determine: 1) whether the adult male transgenic sickle cell mouse (Tg58 × Tg98; TSCM), exhibits the patterns of reproductive endpoints (hypogonadism) characteristic of men with sickle cell disease (SCD) and 2) whether hydroxyurea (HU) exacerbates this condition. In Experiment 1, blood samples were collected from adult age-matched TSCM and ICR mice (ICRM) (N = 10/group) for plasma testosterone measurements. Subsequently, mice were sacrificed, testes excised and weighed and stored spermatozoa recovered for the determination of sperm density, progressive motility and percentage of spermatozoa with normal morphology. In experiment 2, adult male TSCM were orally treated with 25 mg HU/kg body weight/day for 28 or 56 days. Control mice received the vehicle for HU (saline) as described above. At the end of the treatment periods, blood samples were collected for quantification of circulating testosterone. Subsequently, mice were sacrificed, testes and epididymides were recovered and weighed and one testis per mouse was subjected to histopathology. Stored spermatozoa were recovered for the determination of indices of sperm quality mentioned in Experiment 1. Testis weight, stored sperm density, progressive motility, percentage of spermatozoa with normal morphology and plasma testosterone concentrations of TSCM were significantly lower by 40, 65, 40, 69 and 66%, respectively than those of ICRM. These data indicate that adult TSCM used in this study suffered from hypogonadism, characteristically observed among adult male SCD patients. In Experiment 2, HU treatment significantly decreased testis weight on day 28, (0.09 ± 0.004g) that was further decreased on day 56 (0.06 ± 0.003g; treatment x time interaction) compared with controls (day 28, 0.15 ± 0.01g; day 56, 2, 0.16 ± 0.01g). Concomitant with a 52% shrinkage (P<0.001) in area of testes in 56 days of HU treatment, testes from HU-treated TSCM exhibited significant atrophic degeneration in the seminiferous tubules compared with controls. Furthermore, treated TSCM had only Sertoli cells and cell debris remaining in most of the seminiferous tubules in comparison with controls. Leydig cell prominence and hyperplasia were more evident (P<0.05) in the steroidogenic compartments of testes of HU-treated TSCM compared with controls. However, plasma testosterone concentrations were reduced by HU treatment (P<0.05; treatment x time interaction) compared with controls on the two time periods studied. Epididymides from HU-treated TSCM sustained a 25% shrinkage (P<0.05), along with 69 (P<0.005) and 95% reduction (P<0.005), in stored sperm density and sperm progressive motility (treatment x time interaction P<0.05), respectively on day 56 of treatment compared with controls. These data demonstrate that TSCM used in this study exhibited SCD-induced hypogonadism, thus authenticating their use for studying the effect of HU on male reproductive endpoints observed in SCD patients. Secondarily, our data show that HU treatment exacerbated the already SCD-induced hypogonadism to gonadal failure.

New isocratic high-performance liquid chromatographic procedure to assay the anti-sickling compound hydroxyurea in plasma with ultraviolet detection

A new procedure using high-performance liquid chromatography (HPLC) with ultraviolet detection to assay hydroxyurea (HU) levels in plasma has been developed. The drug was isolated from plasma by a direct deproteinization process with sulfosalicylic acid. Following neutralization of the acidic supernatant, an aliquot was loaded onto an Aminex HPX-72S column (300x7.8 mm). Chromatography was performed at 55 degrees C using a mobile phase consisting of acetonitrile-0.025 M ammonium sulfate buffer (pH 8.5) including 0.1% triethylamine, 0.01 M sodium sulfate, and 5 mM sodium heptane sulfonate. The UV absorbance of effluent was monitored at 214 nm. A flow-rate of 0.8 ml/min was used for analyzing HU in both human and mouse plasma. Under these conditions, the drug eluted at 12.6 min. The assay possessed linearity up to 425 microg/ml, with a lower limit of quantitation of 3.32+/-0.0004 microg/ml (mean+/-S.D., n=10). Intra-day and inter-day coefficients of variation were less than 8.5% and 8.7% respectively. Absolute differences were less than 7.4%. The method has been employed in clinical studies and the sensitivity of the assay was shown to be adequate for characterizing the plasma pharmacokinetics of HU in mice. In conclusion, the procedure described herein could be ideally suited for therapeutic monitoring of hydroxyurea.

Direct laser trapping for measuring the behavior of transfused erythrocytes in a sickle cell anemia patient.

Using a laser trap, we have studied the properties of erythrocytes from a sickle cell anemia patient (SCA) after receiving an intravenous blood transfusion, and a normal adult individual carrying normal adult hemoglobin. The hemoglobin type and quantitation assessment was carried out by high performance liquid chromatography (HPLC). We conducted an analysis of the size distributions of the cells. By targeting those erythrocytes in the overlapping regions of size distributions, we have investigated their properties when the cells are trapped and released. The efficacy of the transfusion treatment is also studied by comparing the relative changes in deformation and the relaxation-time of the cells in the two samples.

Hydroxyurea‐induced oxidative damage of normal and sickle cell hemoglobins in vitro: Amelioration by radical scavengers

Hydroxyurea (HU) induces fetal hemoglobin (Hb F) production in patients with sickle cell anemia. The therapeutic dosage of HU used for Hb F induction often elicits myelosuppression, which becomes its major associated complication. We examined the effect of HU on hemoglobin modulation and the role of radical scavengers on these induced changes. In vitro exposure of human blood to various concentrations of HU at predetermined time intervals induced a progressive dose‐dependent oxidation (MetHb formation) of both adult (Hb AA) and sickle (Hb SS) hemoglobins. The oxidative effect of HU on Hb SS was 3 times greater than its effect on Hb AA. Similar but less profound changes were observed in H2O2‐treated samples. Hb F was, however, observed to be relatively resistant to HU‐induced oxidative damage. A substantial protective effect of Hb by α‐tocopherol, ascorbic acid, and D‐mannitol was observed during pretreatment of Hb AA and Hb SS blood samples. Analyses of the hemoglobins and their globin chain components by high‐performance liquid chromatography revealed a considerable protective effect by these free radical scavengers. These results indicate that the HU‐induced damage of hemoglobin and their component globin chains can be reduced by radical scavengers. J. Clin. Lab. Anal. 15:1–7, 2001.

Enriched autonomously replicating sequences in a nuclear matrix-DNA complex isolated from synchronized HeLa cells.

Nucleoids isolated from either synchronized or exponentially growing HeLa cells were digested with restriction enzymes to separate a nuclear matrix-bound DNA component from the rest. Partial libraries were constructed by inserting DNA fragments from both components into a yeast-bacteria plasmid vector. A random sample from these libraries was tested for ARS activity by a standard yeast transformation assay. We found that synchronization for DNA replication results in an enrichment for autonomously replicating sequences in the library constructed with the DNA component bound to the nuclear matrix.

Chromosomal mapping and nucleotide sequence of a human DNA autonomously replicating sequence

A 1.1-kb human DNA fragment (ARSH1) capable of functioning as a putative origin of replication in yeast cells has been characterized both by in situ hybridization to human metaphase chromosomes and by DNA sequencing. Our hybridization studies show a preferential localization of ARSH1 in chromosome regions 1p34-36 and 2q34-37. DNA sequence analysis indicates that in addition to the consensus sequence required for ARS function in yeast cells, nuclear matrix-associated DNA motifs are also present in the 1.1-kb fragment. These results suggest that ARSH1 sequences may serve as points of anchorage to the nuclear matrix for chromosomes 1 and 2.

Hydroxyurea-induced denaturation of normal and sickle cell hemoglobins in vitro

Use of hydroxyurea (HU) to treat sickle cell disease is usually associated with increments in fetal hemoglobin (Hb F) production; however, in vitro studies show that HU may also induce hemoglobin denaturation. Whole blood samples from Hb AA, Hb AS, and Hb SS patients were treated in vitro with 100, 150, 200, 250, and 300 μg/mL HU, incubated at 30°C for up to 12 days, and analyzed by high‐performance liquid chromatography (HPLC). Hb AA levels show decrements of 91 to 14% with 100 μg/mL and 89 to 4% with 150 μg/mL after 12 days; 86 to 2% with 200 μg/mL after 10 days; 86 to 8% with 250 and 300 μg/mL after 8 days. Similar treatment and incubation times for Hb AS whole blood demonstrate that HU equally degrades the A and S components of Hb AS. A comparable approach for Hb SS whole blood samples, using a 300 μg/mL HU treatment, showed a hemoglobin denaturing pattern that went from 93% to 1% after 12 days. Globin chain analysis of these samples by reverse‐phase HPLC showed that the denaturing effects occur mostly on the β‐globin chain. J. Clin. Lab. Anal. 11:208–213, 1997.

HB Inkster [α85(F6)ASP→VAL] Found in a Caucasian male with Polycythemia

’ Comprehensive Sickle Cell Center: Departments of Obstetrics and Gynecology Meharry Medical College, Nashville. TN 3 7208-3599, USA ’ Hemoglobin DNA Laboratory, Departnient of Medicine Medical College of Georgia, Augusta, GA 30912-2100, USA Department of Pediiicitrics, Meharty Medical College Nashville, TN 37208-3599, USA Division of Heniatologv/Oncology, Departntent of Medicine Vnnderhilt University unrl Veterans Adnzinistration Medical Centers Nushvillc, TN 37232-6305, USA 5

Radiographically occult pulmonary metastases from gestational trophoblastic neoplasia

Gestational trophoblastic neoplasia (GTN) is a spectrum of diseases including partial and complete hydatidiform moles, placental site trophoblastic tumor, and choriocarcinoma. One of the most important considerations is recognition of the possibility of GTN after molar pregnancy or even normal pregnancy. It is common practice to use chest x-ray for the detection of pulmonary metastasis. Computed tomography imaging of the lungs is ordered if lung lesions are noted on chest x-rays. However, understanding the limitations of chest x-rays is important for detecting smaller pulmonary lesions. We present a patient with GTN and pulmonary metastasis after having received 2 negative chest x-rays.

Laser Trapping for Single Red Blood Cell (RBC) Ionization and Measurement of Charge

Application of high intensity gradient laser trap for charging a single cell is demonstrated. We used RBCs from normal person (AA hemoglobin) and an individual with sickle cell trait (AC hemoglobin).