Charles D. Lox

The effects of dietary marine fish oils (omega-3 fatty acids) on coagulation profiles in men.

1. This study was undertaken to evaluate the effects of low dose ingestion of omega-3 fatty acids on clotting profiles in healthy men ingesting 3 g of MaxEPA (900 mg omega-3 fatty acids) daily for 30 days. 2. No effect was noted on either platelet aggregation or circulating prostaglandin levels. 3. Significant decreases were noted for total cholesterol and low density lipoprotein. 4. Clotting factor decreases were noted for factors primarily of the intrinsic pathway and several factors which promote fibrinolysis. 5. The data suggests that low level ingestion of marine fish oil has a beneficial effect on lipids and possibly the clotting profiles in healthy men.

Reaction of Human Endothelial Cells to Bovine Hemoglobin Solutions and Tumor Necrosis Factor

Human umbilical vein endothelial cells (HUVEC) were incubated for 24 hours with 0.1 mM or 0.3 mM of: [A] unmodified (U) Hb-FeIIO2; [B] UHb-FeIII; [C] UHb-FeIV-OH; [D] polymerized low molecular weight Hb (< 400 kDa); [E] polymerized high molecular weight Hb (< 1,020 kDa); [F] polymerized low molecular weight Hb + Endotoxin (2.5 EU/mL); [G] rTNF alpha 100 pg/mL; [H] rTNF alpha 400 pg/mL; [I] rTNF alpha 800 pg/mL. The medium of the incubation was tested for LDH (index of cell injury), and for cytokines GM-CSF and IL-1 alpha released by the cells. The data suggests that oxidation status of the iron in the Hb molecule and concentration of Hb play an important role in causing EC injury. The highest toxicity was observed when EC were incubated with 0.1 mM of UHb-FeIV-OH (ferryl-Hb) and no toxicity with 0.3 mM of Hb-FeIII (ferric-Hb). The direct stimulation of EC by Hb for the production of IL-1 was limited, related only to high molecular weight Hb polymers or to Hb+E, however GM-CSF expression was increased by almost all Hb forms. TNF induced dose-related injury (R2 = 0.986), and dose-related release of IL-1 (R2 = 0.977). A different EC reaction was observed on the release of GM-CSF. Intermediate levels of TNF (400 pg/mL) increased the expression of this cytokine, while high levels (800 pg/mL) blocked its release.

CYTOKINES AND PAF RELEASE FROM HUMAN MONOCYTES AND MACROPHAGES: EFFECT OF HEMOGLOBIN AND CONTAMINANTS

Monocytes [M] were isolated from venous blood of healthy volunteers and activated macrophage-leukocytes (Mø-L] were obtained from peritoneal fluid of patients with mild endometriosis. The M were incubated with pyrogen free CELLGRO culture medium [Control], and with 0.2 mM of [A] unmodified bovine hemoglobin (UHb), [B] Hb crosslinked to form polymers with M.W. < 400 kDa (LMWHb), [C] Hb crosslinked to form large polymers (< 1,020 kDa) (HMWHb), and Mø-L additionally with [D] UHb contaminated with endotoxin (Hb+E) (2.5 EU/mL), and [E] UHb contaminated with phospholipids (Hb+PLs). The Mø-L medium of incubation was tested for TNF alpha, IL-1 alpha, IL-6, GM-CSF and PAF after 6 and 24 hours, but M for TNF alpha and GM-CSF at 12, 24 and 36 hours. Mø-L were found more responsive than M colonies. The strongest reaction of Mø-L was to Hb+E, which produced levels of cytokines and PAF higher than Controls (p < 0.001). Hb+PLs induced smaller increases of TNF and IL-6, and a decrease in the levels of IL-1 and GM-CSF. However, the release of PAF was much greater with this Hb than with Hb+E. UHb caused an increase in TNF, as compared to control (p < 0.01). LMWHb generated a similar increase in TNF, but also a decrease in IL-1. Both polymerized Hb forms inhibited expression of GM-CSF. HMWHb induced high levels of TNF, IL-1 and PAF. UHb, LMWHb and HMWHb significantly increase levels of TNF in M cultures after 36 hours of incubation.

Seminal concentrations of total and ionized calcium from men with normal and decreased motility

In this study, sperm motility, velocity, and progression were compared with the total and Ca++ concentrations in the SF from men with normal and decreased motility (less than 60%). No significant difference in SF total calcium content was observed in men with normal and hypomotility. However, a statistically significant decrease in seminal Ca++ was observed in those men with decreased motility, when compared with that of men with normal motility.

Modified Hemoglobin Solution, with Desired Pharmacological Properties, Does not Activate Nuclear Transcription Factor NF-kappa B in Human Vascular Endothelial Cells

The aim of the present study was to evaluate the role of hemoglobin (Hb) and the contribution of chemically modified Hb solutions on the activation of nuclear transcription factor. NF-kappa B, and propagation of oxidative stress within human vascular endothelial cells. The activation of an oxidative stress-sensitive NF-kappa B can be linked with the propagation of an inflammatory state via rapid induction of genes for several pro-inflammatory mediators. Human coronary artery endothelial cells (HCAEC) were cultured on glass coverslips or cell culture plates to confluence. Then, the cells were incubated for up to 18 hours with endothelial basal medium (EBM) supplemented with 5% FBS and test agents in a concentration of 0.1 and 0.2 mmol: 1) unmodified bovine Hb (UHb): 2) modified Hb solution polymerized with glutaraldehyde (GLUT-Hb), and 3) a novel modified Hb solution (Hb-PP-GSH) prepared according to our patented procedure (U.S. Patent No. 5,439,882). The positive control for the NF-kappa B activation study included a treatment of the cells with: I) endotoxin: IL-1; TNF; and H2O2. Results indicate that Hbs pro-oxidant potential was influenced by the type of chemical modification procedure. The GLUT-Hb autoxidation rate, peroxidase-like activity and reactivity with H2O2/ferryl species formation were higher as compared to UHb, by 15%, 35% and 30%, respectively. However, pro-oxidant potential of Hb-PP-GSH was significantly lower than that of UHb (by 22%, 12% and 28%, respectively). The extent of oxidative stress of the HCAECs was found to be the Hb modification-type and concentration dependent. Although the highest endothelial lipid peroxidation and the largest depletion of intracellular GSH was associated with 0.2 mmol of GLUT-Hb, the Hb-PP-GSH did not produce significant changes when compared to the control cells. The UHb generated a moderate oxidative stress to the endothelium. The immunofluorescent and EMSA results indicate a correlation between the type of Hb chemical modification and the induction of NF-kappa B nuclear translocation. We found that GLUT-Hb rapidly activated NF-kappa B and induced nuclear translocation. Treatment of the cells with an increasing amount of UHb leads to the partial nuclear induction of NF-kappa B. However, Hb-PP-GSH did not activate NF-kappa B directly. In this study, the positive control cells treated with endotoxin, IL-1 or TNF demonstrated full nuclear translocations, whereas H2O2 caused only partial induction. In conclusion, nuclear translocation of NF-kappa B by Hb solutions might be dependent on Hbs pro-oxidant potential and extent of Hb-mediated endothelial oxidative stress. Besides the low oxidative potential of Hb-PP-GSH, the observed lack of NF-kappa B activation by this Hb solution can be also related to the anti-inflammatory properties of adenosine which is used in our novel modification procedure. In this study, only the Hb-PP-GSH, cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and combined with reduced glutathiore, was shown to be non-toxic to the endothelium and promises to be an effective free-Hb based blood substitute.

Improved blood substitute: evaluation of its effects on human endothelial cells.

The authors have previously documented that appropriate chemical and pharmacologic modification of the hemoglobin molecule are required to attenuate certain pathophysiologic reactions of the reticuloendothelium. The current study further investigates the molecular responses of human coronary artery endothelial cells to a high concentration (0.4 mmol) of 1) unmodified bovine hemoglobin; and 2) an improved blood substitute that comprises hemoglobin cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione. In this study, the scavenging effect of hemoglobins toward nitric oxide (NO) was evaluated by the measurement of nitrite (NO2-) and nitrate (NO3-) formation. The pro-oxidant effect of hemoglobin on endothelial cells was examined by the measurement of intracellular reduced glutathione, and by monitoring the formation of lipid hydroperoxides and 8-iso prostaglandin F2alpha, a novel potent vasoconstrictor, which is produced by a noncyclooxygenase mechanism involving free radical catalyzed peroxidation of arachidonic acid. The inflammatory reactions of endothelial cells were evaluated by the expression of the adhesion molecule, intracellular adhesion molecule-1, and the activation of nuclear transcription factor, nuclear factor kappaB. In additional, endothelial cell responses were investigated by analysis of intracellular ionized calcium concentrations. Results indicate that unmodified hemoglobin in a concentration of 0.4 mmol/L can aggravate endothelial cell oxidative and inflammatory responses. This hemoglobin produced a significant (p < 0.01) depletion of reduced glutathione, acceleration of lipid peroxidation, and a greater influx of Ca2+. The formation of 8-iso prostaglandin F2alpha increased compared with the control cells (p < 0.01). Unmodified hemoglobin was found to be a potent scavenger of NO, great activator of nuclear factor kappaB, and a stimulator of intracellular adhesion molecule-1 expression. Contrarily, the improved blood substitute did not appear to induce oxidative stress nor to increase the intracellular Ca2+. The concentration of 8-iso prostaglandin F2alpha was similar to that in the control cells, whereas the formation of NO2-/NO3- was much lower (p < 0.05) than in the unmodified hemoglobin group. The effect of an improved blood substitute can be linked with the anti-inflammatory and cytoprotective properties of adenosine, which is used as a cross-linker and surface modifier, and the type of the chemical modification procedure that lowers hemoglobin pro-oxidant potential.

Hyperestrogenism induced by menotropins alone or in conjunction with luprolide acetate in in vitro fertilization cycles: the impact on hemostasis*

OBJECTIVE To evaluate coagulation parameters during IVF cycles with elevated E2. DESIGN Prospective clinical study. SETTING Human volunteers in an IVF clinic. PATIENTS Infertile women undergoing IVF procedures. INTERVENTIONS Coagulation factors were measured in blood along with E2 and P after singular hMG or leuprolide acetate (LA) plus hMG up to 14 days after hCG. MAIN OUTCOME MEASURES Plasma coagulation factors. RESULTS Some coagulation factors were statistically but not clinically elevated after LA-hMG-induced hyperestrogenism. For the most part, this was not correlated with E2. CONCLUSION This study suggests that endogenous E2 increases due to fertility drugs cause a molecular activation of some coagulation factors, which do not result in an increased thrombosis.

The role of dietary molybdenum on estrous activity, fertility, reproduction and molybdenum and copper enzyme activities of female rats

Abstract In this study, we investigated the effect of supplemental molybdenum (Mo) on estrous activity, fertility and reproduction, hepatic xanthine dehydrogenase/oxidase (XDH/OX), sulfite oxidase (SOX) and plasma ceruloplasmin (Cp) in female SD rats. Weanling female rats were assigned to five dietary treatment groups and fed an AIN-76A diet. They were given deionized water containing either no Mo or Mo at 5, 10, 50 and 100 mg/l. Mo significantly prolonged the estrous cycle when fed 10 mg/l or higher. Gestational weight gain was higher for the controls and the 5 mg/l, than for the 10–100 mg/l treatments. Histological data suggested that Mo supplemented at 10 mg/l or higher delayed fetal esophageal development, transfer of fetal hemopoiesis to bone marrow and myelination in the spinal cord. Intrauterine deaths were few, but the rate of fetal resorption increased with supplementation at 10 mg/l or higher. Mean hepatic XDH/XO, SOX and plasma Cu-Cp activity increased with Mo supplementation. This study suggests that supplemental Mo may influence estrous activity and embryogenesis. Hepatic XDH/OX, SOX and plasma ceruloplasmin may be affected differently in gestating and nongestating animals.

Pretreatment Determination of the Serum Urokinase Plasminogen Activator and its Soluble Receptor in Advanced Small-Cell Lung Cancer or Non-Small-Cell Lung Cancer

This study was undertaken to evaluate the circulating urokinase plasminogen activator (uPA), and its soluble receptor (suPAR) in patients with either small-cell lung cancer (SCLC) or non-small-cell lung cancer (NSCLC). Thirty-one male and female patients with stage III or IV NSCLC and 17 with stage III or IV SCLC were compared to 138 age-matched non-smoking controls of both sexes. Before any treatment was undertaken, serum was obtained and evaluated for its content of uPA and suPAR with enzyme-linked immunosorbent assay. The results indicated a wide variation in serum uPA content that was not significant, while extremely significant increases in suPAR were found. The natural physiologic relationship between uPA and its receptor was lost in both SCLC and NSCLC, which could indicate abnormal alterations of uPA suPAR, suggesting these factors aid in the process of invasion and metastasis of lung cancer.

Evidence for the direct inhibition of endothelin-1 secretion by hemoglobin in human endothelial cells.

The actual hemoglobin (Hb) contribution to endothelin-1 (ET-1) production in human umbilical vein endothelial cells (EC) was investigated. Cells were incubated with 0.1 mmol or 0.3 mmol of bovine: 1) unmodified (U) ferrous-Hb; 2) U-ferric-Hb; 3) U-ferryl-Hb; 4) polymerized low molecular weight (m.w.) Hb with chemically modified surface (< 400 kDa); and 5) glutaraldehyde polymerized, high m.w. Hb (< 1020 kDa). The incubation medium was tested at 6 and 24 hr for lactate dehydrogenase (index of cellular injury), and for ET-1 release by the cells. Before radioimmunoassay, the ET-1 was extracted from cell culture medium by a two-step purification procedure: 1) ultrafiltration, and 2) column extraction with C18 cartridges. The data suggested that the oxidation status of Hb and its concentration play an important role in causing EC injury. The highest toxicity was observed when EC were incubated with 0.1 mmol of ferryl-Hb, and there was no toxicity with 0.3 mmol of ferric-Hb. These results indicate that the ferric-Hb and low m.w. polymerized Hb at a concentration of 0.1 mmol did not alter ET-1 synthesis and produced a level similar to that of the control. However, it was found that ferryl-Hb and ferrous-Hb in a concentration of 0.1 mmol significantly reduced ET-1 release. All Hbs at a concentration of 0.3 mmol markedly inhibited the production of ET-1. The greatest decrease in ET-1 levels was produced by ferryl-Hb, and the lowest by ferric-Hb and low m.w. polymerized Hb. The Hbs inhibitory effect was more pronounced at 24 hr of incubation. It was also found that although Hb molecules showed a high degree of cross-reactivity with polyclonal anti ET-1 antibodies, the presence of different Hb solutions in the EC culture medium did not change the immunologic properties of ET-1 peptide. In conclusion, Hb inhibitory activity toward ET-1 production might be related to Hb mediated endothelial oxidative injury.