Protein turnover and energy expenditure increase during exogenous nutrient availability in sickle cell disease.

In the present study, energy expenditure (EE) and rates of whole-body protein, glucose, and lipid metabolism were assessed in 8 African American sickle cell disease (SCD) patients and in 6 healthy African American control subjects during the infusion of amino acids, glucose, and lipid. Whole-body protein, glucose, and lipid kinetics were estimated by using L-[1-(13)C]leucine, D-[6,6-(2)H2]glucose, and [(2)H5]glycerol, respectively. After a 2-h tracer equilibration period and a 0.5-h basal period, nutrients were administered intravenously for 3 h with 16% of the energy as protein, 52% as carbohydrate, and 32% as fat. Breath and blood were collected during the last 30 min of nutrient infusion and EE was measured by indirect calorimetry. EE was 14% greater (P < or = 0.05) in SCD patients [145.0 +/- 3.5 kJ x kg fat-free mass (FFM)(-1) x d(-1)] than in control subjects (126.8 +/- 3.8 kJ x kg FFM(-1) x d(-1)). Whole-body protein breakdown (4.4 +/- 0.4 compared with 3.1 +/- 0.1 mg x kg FFM(-1) x min(-1), P < or = 0.05) and protein synthesis (4.6 +/- 0.4 compared with 3.2 +/- 0.1 g x kg FFM(-1) x min(-1), P < or = 0.05) were 42% and 44% greater, respectively, in the SCD patients than in control subjects, but whole-body amino acid oxidation (0.90 +/- 0.05 compared with 1.03 +/- 0.09 mg x kg FFM(-1) x min(-1)) was not significantly different between the 2 groups. Whole-body glucose and lipid kinetics did not differ significantly between the groups. EE increased in SCD patients during exogenous nutrient availability, and the additional energy required for the accelerated rates of whole-body protein breakdown and synthesis made a significant contribution to the increase in EE. These metabolic aberrations may increase the dietary energy and protein requirements of SCD patients.

Response of luteinizing hormone and follicle-stimulating hormone to different doses of synthetic luteinizing hormone-releasing hormone by intramuscular administration in normal and oligospermic men: preliminary report.

The response of LH and FSH levels to intramuscularly administered synthetic LH-RH was studied in two healthy volunteers and three oligospermic patients. Four tests with 50, 100, 250, and 500 mug of LH-RH, respectively, were carried out on each subject at 8 am; the interval between tests was one week. The serum levels of LH and FSH were determined by radioimmunoassay (double-antibody method) before each injection, and 60, 120, 180, and 240 minutes after each injection. No differences in the basal values of either hormone were observed. In both oligospermic and normal men, maximal responses were obtained with doses between 100 and 250 mug. With 500 mug, levels decreased rather than increased. Maximal peaks occurred between 60 and 180 minutes after injection. In the two normal subjects, the responses of LH and FSH were similar. Two of the three oligospermic patients showed discordant responses. From the results, we can assume that LH-RH doses between 100 and 250 mug should be used as a basis for chronic treatment.

D-leucine-6-luteinizing hormone-releasing hormone ethylamide in the treatment of normogonadotropic oligoasthenospermia.

The effectiveness of D-leucine-6-luteinizing hormone-releasing hormone ethylamide (D-Leu-6-LH-RH-EA) in the treatment of idiopathic normogonadotropic oligoasthenospermia was assessed in 17 patients selected on the basis of at least three previous sperm counts; a history of long-standing infertility; normal serum levels of luteinizing hormone, follicle-stimulating hormone, and testosterone; and lack of any evidence of other pathologic involvement. On testicular biopsy all patients showed hypospermatogenesis or arrest at the spermatid stage with the exception of two with more severe disorders. D-Leu-6-LH-RH-EA- was administered intramuscularly for 90 days at a daily dose of 5 microgram (four patients), 10 microgram (four patients), 20 microgram (five patients), and 200 microgram (four patients). The results showed no significant improvement in the parameters considered (number of spermatozoa per milliliter, percentage of live and motile spermatozoa, and percentage of forwardly progressive spermatozoa). D-Leu-6-LH-RH-EA does not seem promising for the treatment of oligoasthenospermic patients.

Response of luteinizing hormone and follicle-stimulating hormone to different doses of D-leucine-6-LH-RH ethylamide in oligospermic patients.

The rise in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels after intramuscular administration of D-leucine-6-LH-RH ethylamide was studied in seven patients with idiopathic normogonadotropic oligospermia. Four tests with 2.5, 5.0, 10.0, and 20.0 mug of the analog, respectively, were carried out at 8 A.M. on each subject. The interval between tests was 1 week. Serum levels of LH and FSH were determined by radioimmunoassay before (-15 And 0 minutes) and 2, 4, 6, 12, and 14 hours after each injection. No differences in the basal values for either hormone were observed. D-Leucine-6-LH-RH ethylamide produced significant increases in LH and FSH levels for 6 to 12 hours after the injection. Peak values were obtained between 4 and 6 hours. A dose-response relationship for both gonadotropins was observed. The highest average levels of LH and FSH were obtained after injection of 20.0 mug of the drug. A great individual variation in magnitude of response was observed which seemed to be related to the functional reserve of the pituitary. It is suggested that doses of 10.0 to 20.0 mug of D-leucine-6-LH-RH ethlamide/day could be used for chronic treatment in male infertility.

Treatment of idiopathic normogonadotropic oligoasthenospermia with synthetic luteinizing hormone-releasing hormone.

An assessment of the effectiveness of synthetic luteinizing hormone-releasing hormone (LH-RH) in the treatment of idiopathic normogonadotropic oligoasthenospermia was made in 21 subjects selected on the basis of at least three previous sperm counts; a history of long-standing infertility; normal urinary gonadotropin excretion; normal serum levels of luteinizing hormone, follicle-stimulating hormone, and testosterone; and lack of any evidence of other clinical, endocrine, vascular, infectious, and/or urologic involvement. On testicular biopsy, 14 patients showed hypospermatogenesis or arrest up to the spermatid stage (group 1), while the remaining 7 had more severe spermatogenetic disorders (group 2). Libido and/or potency were previously unaltered in all subjects. Five patients in group 1 and five patients in group 2 received intramuscular injection of 100 to 500 mug of LH-RH daily for no less than 60 days (long-term treatment); the remainder received the drug for 30-day periods, with 30-day intervals (short-term treatment). Qualitative and quantitative assessment of the results during and after treatment showed frank improvement in spermatozoal concentration, vitality, and motility in group 1 patients, particularly in those undergoing long-term treatment (three of five patients under these conditions were able to impregnate their wives). The long-term regimen also resulted in predomantly qualitative improvements in group 2 (one of five patients impregnated his wife). The short-term regimen was less effective. Improvements under either regimen usually occurred at the end of, or after, treatment. Six of seven patients in group 1 who were followed for a long period retained values above preliminary values for 90 to 150 days after the drug was discontinued. Increased libido and potency were spontaneously reported by 15 patients, 20 to 30 days after the start of administration of synthetic LH-RH.

Luteinizing hormone (LH), follicle-stimulating hormone, and testosterone responses to consecutive injections of D-leucine-6-LH-releasing hormone ethylamide in normal men.

Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) responses to three consecutive intravenous injections of D-Leu-6-LH-releasing hormone ethylamide (D-Leu-6-LH-RH-EA) at 3-hour intervals were studied in six healthy, fertile, male volunteers 34.2+/-1.6 years of age. Each man received three injections of 20 microng of D-Leu-6-LH-RH-EA at 6:00 A.M., 9:00 A.M., and 12:00 noon, respectively. Blood samples were obtained before the first injection and at 1, 2, 3 (before the second injection), 4, 5, 6 (before the third injection), 7, 8 and 9 hours after the beginning of the test. Serum levels of LH, FSH, and T were determined by radioimmunoassay with the double-antibody technique. The response to the first injection of D-Leu-6-LH-RH-EA confirmed the longer duration of the stimulation of LH and FSH release caused by this compound as compared with that caused by LH-RH. Serum T levels rose significantly, almost paralleling the variations experienced with gonadotropins. The second injection caused a slight increase in LH and T responses in relation to the first injection. Two and three hours after administration, the third stimulus resulted in hormone levels lower than those obtained with the first two injections. Possible explanations for this finding might be a reduction of pituitary responsiveness as a result of multiple stimulation with D-Leu-6-LH-RH-EA, or spontaneous circadian variation of the pituitary response, or a combination of factors.

Reference ranges for hemoglobin variants by HPLC in African Americans.

High performance liquid chromatography (HPLC) demonstrated advantages over conventional procedures employed in newborn and adult hemoglobinopathy screening programs for the identification of Hb variants has promoted the need to reassess our knowledge of hemoglobin reference ranges as it relates to HPLC quantitation. In this study, the HPLC hemoglobin reference ranges derived from 200 normal African American adults are expressed as follows: Hb A mean 93.6 percent (s.d. 1.3, ranges 89.8 to 95.2), Hb A1 mean 2.0 percent (s.d. 0.6, ranges 0.8 to 5.2), Hb F mean 3.2 percent (s.d. 0.7, ranges 1.7 to 5.3) and Hb A2 mean 1.2 percent (s.d. 0.4, ranges 0.5 to 3.4); while the HPLC results for normal newborns and babies (n = 99) in the African American population fluctuates from Hb F mean 82.0 percent (s.d. 7.7, range 66.6 to 89.9) and Hb A mean 19.0 percent (s.d. 7.7, ranges 10.1 to 33.4) at 4 days to a mean of 15. percent (s.d. 4.8, range 9.3 to 22.8) for Hb F and a mean of 85.0 percent (s.d. 5.1, ranges 76.4 to 90.7) for Hb A at 300 days after birth. In case of the most common hemoglobin variants for this population, it has been shown that the A/S and A/C ratios for adults (Hb AS, Hb AC) and newborns (Hb FAS, and FAC) remained within the 1.5 (range 1.0 to 2.2) limits regardless of age group. Application of these HPLC ranges to confront other abnormalities will prove most useful during blood screening processes.

Hemoglobin variant detection from dried blood specimens by high performance liquid chromatography.

The convenience of dried blood filter paper specimens for genetic screening programs has prompted us to test the stability of these specimens for hemoglobin identification by cation exchange high performance liquid chromatography. This report shows that identification of Hb AA, Hb AF, Hb AS, Hb FAS, Hb AJ, Hb FJ, Hb EF, and Hb SS can be achieved by high performance liquid chromatography even after six weeks of storage at room temperature. Also, accurate hemoglobin quantitation can be obtained from the same samples within three weeks of storage at room temperature. The combination of dried blood samples and high performance liquid chromatography provides an accurate system to screen for hemoglobinopathies, even after long periods of sample storage at ambient conditions.

Chromatographic measurements of hemoglobin A2 in blood samples that contain sickle hemoglobin.

In the sickle cell syndromes, Hb A2 measurements aid in the differential diagnosis of sickle cell anemia from sickle-beta-thalassemia. The purpose of this study is to assess the Hb A2 levels in samples containing sickle hemoglobin (Hb S) by the use of an automated high performance liquid chromatography system (HPLC-Variant beta-thalassemia Short Program). The blood samples analyzed were from individuals of African descent living in the state of Tennessee who had either sickle cell trait (Hb AS), sickle cell disease (Hb SS), or sickle cell-hemoglobin C disease (Hb SC). Interestingly, the Hb A2 levels determined by HPLC were found elevated in samples containing Hb S. The Hb A2 mean in Hb AS samples (n=146) is 4.09% (SD +/- 0.42, range 2.20 to 5.20%); in Hb SS samples (n=33) it is 3.90% (SD +/- 1.08, range 0.60 to 5.90%); and in Hb SC samples (n=27) it is 4.46% (SD +/- 0.70, range 2.30 to 5.91%). The Hb A2 mean by HPLC in normal individuals (Hb AA, n=70) is 2.57% (SD +/- 0.25, range 2.1 to 3.0%), and the Hb A2 range in beta-thalassemia carriers is 4 to 9%. Our results show that the Hb A2 levels in Hb S-containing samples partially overlap with those expected from beta-thalassemia carriers. The hemoglobinopathy laboratory should be aware of this apparent elevation in Hb A2 levels determined by HPLC in individuals carrying Hb S. Other factors, such as family history and clinical symptoms, should be taken into account before a diagnosis of sickle cell trait, sickle-beta-thalassemia, or sickle cell anemia is made.

Severe anemia: a risk factor for glomerular injury in sickle cell disease.

Approximately 15% to 20% of patients with sickle cell disease have proteinuria. Proteinuria, particularly albuminuria, is the hallmark of glomerular injury. This study examines risk factors for glomerular injury as indicated by urinary albumin excretion (UAE) 30 microgram/minute, directly related to sickle cell disease. A total of seven patients were enrolled between September 1992 and March 1993. Fasting blood chemistries, complete blood cell count, 24-hour urine for protein and creatinine clearance, and glomerular filtration rate determined by 125 I-iothalamate were obtained for each patient. The results indicated that the lower the hematocrit, the higher the UAE rate. Low hematocrits have served as a protective mechanism in sickle cell disease by reducing blood viscosity and thus decreasing the number of vaso-occlusive crises. However, severe anemia appears to have an indirect adverse effect on the kidney in sickle cell disease.

Sickle cell disease with complicated influenza B virus infection.

The Committee on Infectious Diseases of the American Academy of Pediatrics, and the Advisory Committee on Immunization Practices of the Center for Disease Control for many years have recommended the routine use of influenza vaccine in various hemoglobinopathies including sickle cell disease. This recommendation, however, has not been included in the patient care protocols of the Comprehensive Sickle Cell Centers program of NIHLB. Most clinicians have not used yearly influenza vaccine for their patients with sickle cell disease. This article reports a case of a 5-year-old boy with sickle cell disease who had not received influenza vaccine. He developed pneumonitis and acute myositis during a serologically confirmed influenza B virus infection. The incapacitating and protracted course of his illness presented diagnostic and management problems. His case strongly supports the recommendation of the two infectious disease committees.

Report of 10 operations under local cerebral hypothermia.

A method of local cerebral hypothermia with circulatory arrest of one or more of the major vessels of the neck is described. Ten clinical cases have been operated upon, and much has been learned of the operative difficulties including an increased operating time and a high complication rate. There were four post-operative deaths, in one (case 1) there was some evidence that the technique protected the perfused part of the brain from anoxic damage. In case 9 the method itself caused particularly bad operating conditions and eventually the patient died. Case 2 and case 4, like case 1, were patients who were severely ill and who might well have died whatever technique had been used in operating upon them. Of the survivors, one patient (case 3) had a post-operative intracerebral clot which developed during closure and another (case 6) had an intracerebral clot which required removal after 24 hours. Another patient (case 8) had some delay in return to full mental function. In the light of this experience we cannot regard the method in its present form as satisfactory for general use. In our view, its further development requires the discovery of a more effective means of neutralizing the anticoagulants, or doing without anticoagulants altogether. It is also necessary to develop a method of monitoring parts of the brain distant from those directly perfused to give warning of threatened anoxia. Until such time as these problems can be solved we have returned to other procedures but are publishing our results in the hope that other workers will be able to improve upon them.

Pentoxifylline (BL 191) by oral administration in the treatment of asthenozoospermia.

The aim of this trial was to study the effects on semen of pentoxifylline administered chronically per os to patients with normogonadotropic asthenozoospermia. Fifteen infertile men (age 22 to 44 years) were incorporated. Each subject was submitted to a treatment with pentoxifylline per os at a dose of 1,200 mg per day during no less than four months (x plus or minus SE 6.6 plus or minus 0.7 months). Assessment of the results was performed by serial spermatograms performed during and up to three months after treatment. The results showed a significant improvement of the percentages of forwardly progressive spermatozoa and of live and motile spermatozoa. Five of the patients achieved a normalization of semen (frank improvement) and seven a significant improvement in comparison to the initial values. Pregnancy was obtained by two patients. Pentoxifylline could be a useful alternative for the treatment of patients with normogonadotropic asthenozoospermia.

Non-chromatographic radioimmunoassay procedure for urinary aldosterone.

We describe a sensitive, specific, and simple procedure for measuring aldosterone in human urine, which requires no chromatographic purification before quantification by radioimmunoassay but does include hydrolysis and extraction steps. Rabbit anti-aldosterone serum is sued, generated against aldosterone-18,21-dihemisuccinate coupled to human serum albumin. The antibody cross reacted little with other structurally related steroids that are in human urine. Our procedure was validated by comparing values for urinary aldosterone in human urine, with and without preliminary purification by chromatography on either paper (y = 0.92x + 2.9; r = 0.99; p less than 0.01) or (Sephadex LH-20) column (y = 0.98x + 0.6; r = 0.99; p less than 0.01). Values by our procedure also correlated well (y = 1.03x - 0.8; r = 0.99; p less than 0.01) with those obtained with use of a validated commercial "kit" for urinary aldosterine. All reagents for the proposed method are available commercially.

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 micrograms/mL HU, incubated at 30 degrees 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 micrograms/mL and 89 to 4% with 150 micrograms/mL after 12 days; 86 to 2% with 200 micrograms/mL after 10 days; 86 to 8% with 250 and 300 micrograms/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 micrograms/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 beta-globin chain.

Effect of the phosphodiesterase inhibitor Pentoxyfylline on human sperm motility.

Tyrode fluid and Tyrode fluid plus Pentoxifylline were individually added to aliquots of semen samples obtained from 6 normal men and 6 infertile patients considered to have idiopathic normogonadotropic oligoasthenozoospermia. Pentoxifylline was added to final concentrations of 0.15, 0.30 and 0.60 mM. One aliquot with no addition served as control. Samples were incubated in 37 degrees C and observed by light microscopy at 30 minutes and at 1, 2 and 4 hours after obtaining the material. At observation time, semen quality was evaluated by determining the percentages of forwardly progressive spermatozoa, slowly progressive spermatozoa, "in situ" motile spermatozoa, live and non-motile spermatozoa and dead spermatozoa. Results reported included only the first and last category. Tyrode fluid did not affect significantly the motility and the duration of activity of spermatozoa. Ejaculated human spermatozoa both from normal and asthenozoospermic men added the Pentoxifylline at 0.30 and 0.60 mM showed a longer lasting activity than those of control semen and semen added only with Tyrode fluid.

Tiazofurin-induced autosecretion of IL-6 and hemoglobin production in K562 human leukemia cells.

Previous reports have established the synthesis of interleukin-6 (IL-6) and IL-6 receptors (IL-6R) in several human leukemia cells and found that IL-6 and the IL-6R could be expressed in cell lines with erythroid/megakaryocytic features. IL-6 is a pleiotropic cytokine involved in megakaryocytic differentiation. The finding that endogenous IL-6 levels in serum increased after 5-fluorouracil (5-FU) treatment suggests that IL-6 may play some role in the recovery of hematopoietic systems. This observation may assist the understanding of erythroid regeneration caused by antineoplastic agents such as tiazofurin. Tiazofurin inhibits the activity of IMP dehydrogenase. Its exposure to K562 cells at 10 microM tiazofurin stimulates erythroid differentiation. Stimulation of cells with tiazofurin gave a significant increase in IL-6 production. Its levels were quadrupled after 2 days of culture. Tiazofurin also caused a trivial reduction in the percentage of cells with the IL-6R. This evidence implies that tiazofurin produced no significant effect on the IL-6R. Tiazofurin also increased the percentage of benzidine-positive cells representing hemoglobin production, confirmed by GpA expression. We concluded that IL-6 is rate limiting in regard to hemoglobin production and that IL-3 could be used for clinical benefit to stimulate erythropoiesis and synergize with tiazofurin.