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1-Deamino-8 D-arginine vasopressin (DDAVP) has been used effectively to normalize the bleeding time in various hemostatic disorders. In von Willebrand disease the reduction in bleeding time is due to the preferential release of large multimers of von Willebrand factor from endothelial cells. However, since the bleeding time correction in patients with uremia and liver disease is independent of the release of von Willebrand antigen and activity, other mechanisms of action of DDAVP need to be considered. Endothelial cells generate several thromborepellant factors including 13-hydroxyoctadecadienoic acid (13-HODE), an inhibitor of platelet adhesion to subendothelium. Using cultured fetal bovine aortic endothelial cells (FBAECs), we have investigated whether DDAVP modulates the production of 13-HODE. We have demonstrated that 14C-linoleic acid labeled FBAECs release several oxygenated derivatives of linoleic acid following a 120 min incubation in the presence of serum. One of these products was identified by chromatographic procedures as 13-HODE. The production of 13-HODE was decreased significantly by DDAVP (1-100 ng/ml) with maximal reduction (approx. 25%) seen at 1 ng/ml of DDAVP. While vehicle treated control FBAECs generated 6780 +/- 690 cpm of 13-HODE per 10(6) cells (mean +/- SE, n = 8), DDAVP treated FBAECs produced 4950 +/- 310 (P < 0.01), 5390 +/- 390 (P < 0.01), and 5720 +/- 410 cpm (P < 0.05) of 13-HODE at 1, 10, and 100 ng/ml DDAVP respectively. Our findings of a decrease in 13-HODE would explain the previously observed morphologic changes of increased platelet adhesion to subendothelium following DDAVP infusion and contributes to our understanding of the mode of action of this therapeutic agent in hemostatic disorders.

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Conversion of arachidonic acid to eicosanoids by kitten retinae was investigated to evaluate whether the pattern of kitten retinal eicosanoids simulates that found in the human and other animal species. Freshly isolated kitten retinae were incubated with 20 microM radiolabeled arachidonic acid, and the metabolites were analysed by reverse phase-high pressure liquid chromatography, thin-layer chromatography and gas chromatography-mass spectroscopy. Kitten retinal tissues converted arachidonic acid into prostaglandins (PGs), thromboxane (Tx) and hydroxyeicosatetraenoic acids (HETEs). The major eicosanoid identified was 6kPGF1 alpha--the stable non-enzymatic hydrolysis product of prostacyclin. Other eicosanoids identified included TxB2, PGE2, PGF2 alpha, 12-hydroxy-heptadecatrienoic acid, 12-HETE, and 15-HETE. The spectrum of kitten retinal cyclooxygenase metabolites is similar to those obtained from bovine retina and human retinal vascular endothelium with prostacyclin being the major cyclooxygenase metabolite produced.

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The chronic phase of O2-induced retinopathy is characterized by retinal neovascularization. We have previously demonstrated that 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), a product of white cells, is proangiogenic. In this study, kittens exposed to in vivo hyperoxia produced increased amounts of 15-HETE. Nine litters of 30 kittens (aged 6-8 days) were used. Control kittens were left in room air; hyperoxic kittens were placed in 80% oxygen for 48 h; recovery kittens were returned to room air for 24 h following hyperoxic exposure. Following treatments, the animals were sacrified, and blood was evaluated for 15-HETE. Stimulated serum 15-HETE levels were assayed by high-performance liquid chromatography and GC-selected ion monitoring. While controls produced 0.48 +/- 0.16 (SE) nmol/ml of 15-HETE, values in the hyperoxic and recovery animals were increased at 0.7 +/0 0.2 and 0.68 +/- 0.15 nmol/ml (p less than 0.05 and p = 0.05, respectively). Increased production of this proangiogenic metabolite by WBCs (which can migrate out of blood vessels to set up extravascular angiogenic foci) may play a role in the genesis of the neovascularization process that occurs in response to oxygen-induced injury.

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When carbacyclin (5E-6a-carba-prostaglandin I2) was added to the maternal afferent circulation of in vitro perfused placentae from normal term pregnancies, relatively little carbacyclin was found in either the maternal or fetal efferent circulations. When carbacyclin was added to the perfusate at 1.0 microM, the peak level in the maternal effluent was only 0.06 microM and in the fetal effluent, 0.026 microM. When infused at 10 microM, 0.77 microM carbacyclin was measured in the maternal effluent and 0.13 in the fetal effluent. These findings demonstrate that carbacyclin is transferred across the placenta from the maternal side to the fetal, but that the net transfer is small. The assay procedure employed HPLC resolution, followed by capillary gas chromatography and selected ion monitoring using PGB as an internal standard. The low levels of carbacyclin detected in the effluents did not result from poor recovery in the analyses. When carbacyclin was added to maternal or fetal effluents at 1 microM, the recovery averaged 85.4 +/- 14.1% (SD); at 10 microM recovery averaged 97.3 +/- 4.2%. Much of the loss of carbacyclin on passage through placental circulation resulted from metabolism. Extracts of both fetal and maternal effluents from placenta perfused with carbacyclin contained a component which on reverse phase HPLC appeared less polar than carbacyclin. When analyzed by GC/MS as the methyl ester-trimethylsilyl ether, this component had a mass spectrum expected for 15-dehydro-carbacyclin. When the presumed metabolite was further converted to the methoxime, the mass spectrum was identical to published spectra for that derivative of 15-dehydro-carbacyclin. When extracts of fetal effluents were analyzed for 15-dehydro-carbacyclin metabolite as well as carbacyclin, it appeared that the metabolite accounted for the majority of the carbacyclin recovered. Most of the metabolite was apparently not formed in the fetal circulation, since when carbacyclin was added to the fetal afferent circulation, little 15-dehydro-carbacyclin was observed in either efferent fluid, and most of the perfused carbacyclin was recovered unaltered in the fetal effluent.

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The effect of 13-hydroxyoctadeca-9,11-dienoic acid (13-HODE), a major lipoxygenase product of endothelial cell linoleic acid metabolism on thrombin-induced platelet thromboxane B2 (TxB2), and 12-hydroxyeico-satetraenoic acid (12-HETE) production was evaluated. 13-HODE inhibited thrombin-induced TxB2 production in human platelets in a concentration-dependent manner. At concentrations of 10 and 30 microM, 13-HODE inhibited TxB2 production by 28 +/- 8% (1SE, n = 5; P less than 0.05) and 48 +/- 6% (P less than 0.01) respectively. 13-HODE (30 microM) also inhibited the production of platelet hydroxyheptadecatrienoic acid (38 +/- 5%, P less than 0.01). A concomitant stimulation of 12-HETE production by 13-HODE was observed (25 +/- 5% and 49 +/- 22% over control values at 10 and 30 microM respectively, P less than 0.01). Our results demonstrate a differential effect of 13-HODE on thrombin stimulated platelet cyclooxygenase and lipoxygenase metabolites.

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Prolongation of bleeding time has been previously observed in hemophilia, although no cause has been elucidated. We measured bleeding time, platelet aggregation, nucleotide release, and thromboxane B2 (TXB2), plasma 6-keto-PGF 1 alpha, platelet-associated IgG (PAIgG), and circulating immune complexes in 31 unselected patients with severe hemophilia A and in 17 controls. In 85% of patients with hemophilia A, the bleeding time was greater than 2 SD above the control level (greater than 8 minutes). Sixty-six percent of patients with hemophilia A had circulating immune complexes, and there was a striking relationship between the presence of these complexes and prolonged bleeding time. Plasma 6-keto-PGF 1 alpha levels were significantly elevated in the patient group, and correlated with bleeding time changes. Platelet aggregation and nucleotide release were normal in the patients with hemophilia, although reduced platelet TXB2 biosynthesis was noted in 26%. No correlation was demonstrated between bleeding time and impairment of platelet TXB2 formation. Seventy-two percent of the patients with hemophilia A had elevated levels of PAIgG, and an inverse relationship between PAIgG and platelet count was observed. No relationship was noted between platelet count and bleeding time. This study indicates that the majority of patients with hemophilia A have prolonged bleeding times. The close correlation between bleeding time, plasma 6-keto-PGF 1 alpha levels, and the presence of circulating immune complexes suggests a role for immune complex-mediated defects in vascular function as the basis for bleeding time prolongation.

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Marked platelet hyperaggregability to adenosine diphosphate, epinephrine, and collagen was demonstrated in two children with vitamin E deficiency, with complete reversal following E supplementation. No clinical thrombotic tendency was observed during the E-deficient state. The action of vitamin E in the schema of platelet arachidonate peroxidation appears to be at the step of phosphilpase A activation, or the conversion of arachidonic acid into the cyclic endoperoxides, since the peroxidation product malonaldehyde was increased during the E-deficient state with normalization following E sufficiency.

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Inability to absorb oral iron is believed to be an extremely rare cause of therapeutic failure in the treatment of iron deficiency anemia. Six patients who had failed to respond to oral iron therapy were studied by a simple oral absorption test and contrasted with 25 patients with untreated iron deficiency anemia and 10 normal subjects. All six of the patients who were therapeutic failures demonstrated impaired iron absorption in the absence of other clinical evidence of gastrointestinal disease. In the 25 newly diagnosed patients with iron deficiency. 24 demonstrated elevated iron absorptions while 10 ironreplete normal subjects had minimal elevations in their serum iron values following the administration of the test dose of 1 mg of elemental iron per kilogram. When the therapeutic failures were treated with parenteral iron, all had a therapeutic response. In addition, after treatment the impaired absorption of iron improved transiently. All children who absorbed iron readily responded to oral iron therapy.

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Chronic iron deficiency in rats resulted in decreased MAO activity both in vitro and in vivo. Since MAO is an important enzyme in inactivation of catecholamines, urinary excretion of DA, NE, E, MN-NMN, and VMA was measured in 24-hour samples from 11 iron-deficient children before and after treatment with intramuscular iron. Pretreatment NE excretion was abnormally high and returned to normal (P=0.001) within one week of therapy. VMA excretion also was higher before than after treatment (P greater than 0.05), but most values were within the normal range for healthy children of comparable size. There was no significant difference between DA, E, and MN-NMN excretion before and after iron therapy. Anemic, non-iron-deficient children had normal urinary NE, E, and VMA excretion before and after transfusion. These findings suggest that the irritability, lack of attentiveness, and low performance scores of iron-deficient children may be related to alterations in catecholamine metabolic pathways secondary to dependence of MAO on adequate iron stores.

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