Heparin enhances endothelial cell von Willebrand factor content by growth factor dependent mechanisms.

Von Willebrand factor (vWf), a multimeric adhesive glycoprotein synthesized, stored, and secreted in megakaryocytes and endothelial cells, is normally found in plasma, platelets and subendothelium. While many substances mediate the release of vWf from endothelial cells, factors that enhance vWf synthesis and partitioning to its regulated pathway are currently unknown. We studied the effect of pharmacologic doses of heparin on the vWf content of endothelial cells. After a lag of 8 h and in the presence of crude or purified growth factor, heparin at doses between 0.25 and 2 U (1.4-11 micrograms)/ml, increased the content of high molecular weight vWf. The increased amounts of vWf were localized to Weibel-Palade bodies and extracellular matrix. Lower molecular weight highly sulfated heparin or heparin-like compounds were most active in growth factor dependent endothelial cell vWf expression. There was no clear importance of polysaccharide sequence or protein core.

Method of administration influences the serum cholesterol-lowering effect of psyllium.

To determine whether psyllium must be mixed with food to lower serum cholesterol, 18 modestly hypercholesterolemic subjects were studied for three 2-wk periods, in random order, separated by a 2-wk return to a National Cholesterol Education Program Step 2 diet. Compared with values for subjects consuming control wheat-bran cereal (63 g/d), after 2 wk of 54 g psyllium-enriched cereal/d containing 7.3 g psyllium, serum total, LDL, and HDL cholesterol, respectively, were reduced by 8% (6.15 +/- 0.15 vs 6.71 +/- 0.19 mmol/L, P < 0.01), 11% (4.24 +/- 0.15 vs 4.78 +/- 0.19 mmol/L, P < 0.02), and 7% (0.99 +/- 0.05 vs 1.07 +/- 0.05 mmol/L, P < 0.01). When 7.6 g of the same type of psyllium as in the test cereal was taken between meals, serum total (6.50 +/- 0.19 mmol/L), LDL (4.50 +/- 0.21 mmol/L), and HDL (1.06 +/- 0.06 mmol/L) cholesterol were no different from control values, and total cholesterol was greater than after psyllium cereal (P < 0.05). We conclude that psyllium must be mixed with foods to have the maximum effect on serum cholesterol.

Psyllium reduces blood lipids in men and women with hyperlipidemia.

To see if a modest amount of soluble fiber reduced blood lipids in subjects with hyperlipidemia who were on a low-fat diet, 42 subjects (21 men, 21 women) consuming an American Heart Association step 2 diet took two servings of breakfast cereal daily for two 2-week periods in a randomized crossover trial. There were two types of test cereals, each providing 6.7 g psyllium fiber daily, and two types of wheat bran control cereals, matched for available carbohydrate and total fiber. Half the subjects tested each type of cereal, and the results were pooled because the psyllium cereals had similar effects on serum cholesterol levels. Comparing values at the end of 2 weeks, psyllium reduced serum total (6.33 +/- 0.12 mmol/L versus 6.76 +/- 0.12 mmol/L, p < 0.001), low-density lipoprotein (LDL; 4.36 +/- 0.11 mmol/L versus 4.73 +/- 0.12 mmol/L, p < 0.001) and high-density lipoprotein cholesterol levels (HDL; 1.10 +/- 0.05 mmol/L versus 1.14 +/- 0.05 mmol/L, p < 0.05) and the LDL/HDL cholesterol ratio (4.27 +/- 0.20 versus 4.48 +/- 0.22, p < 0.02) with no effect on triglycerides. There was no significant interaction between the effects of treatment and sex for any of the blood lipid variables. Women tended to have greater decreases in total, LDL, and HDL cholesterol levels than men, but the percent decrease in LDL/HDL ratio on psyllium was similar in men, 4.9%, and women, 4.7%. It is concluded that 6.7 g of psyllium fiber daily, with a low-fat diet, reduces serum cholesterol levels in both men and women with hyperlipidemia.

Phase II metabolism of warfarin in primary culture of adult rat hepatocytes.

We used adult rat hepatocytes in primary culture (HPC) as a model system to study the hepatic phase II metabolism of the anticoagulant warfarin. Hepatocytes were isolated by a collagenase perfusion technique and maintained for 24 hr in Waymouth's medium containing 0.1 mM (R)-warfarin. When HPC medium was analyzed by reverse phase high performance liquid chromatography with diode-array detection, 4'-, 6-, and 7-hydroxywarfarin were identified. Several putative conjugates were observed eluting between 13 and 18 min. Treatment of hepatocyte medium with beta-glucuronidase and sulfatase resulted in the loss of five putative conjugates and concomitant increases in 4'-, 6-, and 7-hydroxywarfarin and warfarin, suggesting that these metabolites and warfarin were conjugated. Use of the beta-glucuronidase inhibitor saccharic acid 1,4-lactone enabled the determination of the relative extents of conjugation of each metabolite by glucuronic acid and sulfate. Glucuronidation was the predominant pathway for 4'-hydroxywarfarin, whereas 6-hydroxywarfarin and warfarin occurred mainly as sulfate conjugates. In contrast, 7-hydroxywarfarin was converted to both glucuronide and sulfate conjugates. Exposure of HPC to phenobarbital resulted in a decrease in cytochrome P-450-mediated production of hydroxylated warfarin metabolites; however, an increase in the production of 8-hydroxywarfarin was observed when HPC were exposed to beta-naphthoflavone. Unique conjugation patterns were found when hydroxylated warfarins were substituted for warfarin in HPC medium. Both 7- and 8-hydroxywarfarin were converted to one sulfate and two glucuronide conjugates, whereas 4'-hydroxywarfarin was converted to a single glucuronide conjugate. A spectral library of these conjugates was used to identify the major conjugates of warfarin formed by rat HPC.

A heparin-like anticoagulant as part of global abnormalities of plasma glycosaminoglycans in a patient with transitional cell carcinoma.

A patient is described in whom a circulating heparin-like anticoagulant developed during the terminal course of metastatic transitional cell carcinoma. The anticoagulant, which was identified as heparan sulfate, was the clinical sign of global abnormalities in the patient's plasma glycosaminoglycans. Subsequent analysis disclosed increased amounts of chondroitin sulfate as well as heparan sulfate. In addition, the charge density and molecular weight of the patient glycosaminoglycans and their organization into proteoglycans differed significantly from glycosaminoglycans isolated from normal plasma samples.

Effect of method of administration of psyllium on glycemic response and carbohydrate digestibility.

To determine whether there was any advantage to taking a soluble fiber supplement separate from food, as opposed to incorporated into a food, we used psyllium as a model, either taken in water just before a flaked bran cereal test meal, sprinkled on top of the cereal, or actually incorporated into the flake. In normal subjects, psyllium reduced the glycemic response when sprinkled onto or incorporated into the cereal, but not when taken before the cereal. Varying the amount of psyllium incorporated into the cereal from 0 to 20% resulted in a linear dose-dependent reduction of the glycemic index (GI) (GI = 101 -2.2 x % psyllium; r = 0.950; p less than 0.002). In subjects with diabetes, the blood-glucose-lowering effect of the psyllium flake cereal was similar to that in normal subjects. Mixing psyllium with the cereal or incorporating it into the cereal reduced the rate of digestion of bran flakes in vitro but was not associated with increased breath hydrogen levels in vivo as an index of rapid colonic fermentation. The bran flakes with psyllium incorporated was rated as no less palatable than the bran flakes cereal alone, and significantly more palatable (p less than 0.05) than taking psyllium in water before the cereal or sprinkling psyllium onto the cereal. These studies confirm earlier reports that viscous fibers must be intimately mixed with the food to have the effect of reducing blood glucose responses, and that the mechanism of action relates to a reduced rate of digestion rather than carbohydrate malabsorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparin-like anticoagulant associated with systemic candidiasis.

A 15 year old girl with aplastic anemia developed a heparin-like anticoagulant during the course of systemic candidiasis. This was initially detected in the laboratory by an elevation of the thrombin clotting time which corrected with toluidine blue but not by mixing with normal plasma. In vivo and in vitro the anticoagulant was remarkably resistant to neutralization by protamine sulfate. Nevertheless, its heparin-like nature was confirmed by its sensitivity to heparinase and its dependence on antithrombin III.

Dental health status and treatment needs of elderly residents of Edmonton, Alberta.

The oral health of 140 independent elderly volunteers, selected from different sections of Edmonton, Alberta, was assessed in conjunction with their nutritional and medical status, in an attempt to evaluate the dental needs of this population. Dental caries was assessed using WHO standards and the CPITN was used in assessing periodontal and gingival health needs. The mean age of the participants was 70.9 +/- 2.6 years. The average time since they had last seen a dentist was 3.0 +/- 6.2 years. Twenty-six percent of the group were edentulous and wore full dentures. An evaluation of denture hygiene and retention showed that 53 per cent of all maxillary and 57 per cent of all mandibular appliances exhibited poor hygiene; 33 per cent and 54 per cent, respectively, had problems with retention and/or occlusion. Only 12 per cent of the group exhibited any sort of mucosal pathology, all of which was related to ill fitting dentures. The total population had a mean of 15.0 +/- 11.1 teeth, only 0.5 of these were decayed, while 8.9 were restored. CPITN scores taken from a total of 511 sextants with standing teeth showed that 16 per cent of the group exhibited bleeding upon probing, 29 per cent had calculus, 13 per cent exhibited pocketing, while 3.6 per cent had deep pockets. Sixty-seven per cent of the population required dental treatment, none of whom needed emergency intervention. Over 49 per cent of the dentate population could benefit from prophylaxis; 16 per cent required more definitive periodontal treatment. Forty-five per cent of the denture population required treatment.

The effect of molecular weight on heparin binding to platelets.

Low molecular weight heparin is reported to be less reactive with platelets than larger heparins. We probed the molecular basis for this pattern of reactivity by characterizing the saturable platelet binding of [3H]heparin in plasma using heparins of different molecular weights (Mr approximately 3000, approximately 5000, approximately 10,000, approximately 15,000). Binding affinity increased with increasing molecular weight, as expressed by decreasing apparent dissociation constants (Kdapp approximately 1.3 microM for Mr approximately 3000, to Kdapp approximately 0.31 microM for Mr approximately 15,000). After adjusting for the effect of antithrombin III in the plasma, true dissociation constants (Kd) could be calculated and these showed the same trend with molecular weight (Kd approximately 1.1 microM for Mr approximately 3000 to Kd approximately 0.096 microM for Mr approximately 15,000). Platelet binding capacity for the different heparin fractions also increased with molecular weight, although this correlation appeared to lessen with the largest species. Heparin antithrombin III affinity was shown not to affect heparin binding to platelets. We propose a model in which heparin binding to platelets is mediated by charge interaction. Larger molecules with more charge bind with greater affinity and to sites with a broader range of electronegativity than do smaller, less

Heparin binding to resting and activated platelets.

Heparin inhibits platelet function and can cause thrombocytopenia. In an effort to understand these phenomena, we have measured the binding of (3H)-heparin to resting and stimulated platelets. In platelet-rich plasma, a single class of saturable heparin binding sites was observed (apparent dissociation constant [kd] approximately 0.55 microgram/mL, R approximately 0.059 microgram/10(9) cells). In gel-filtered platelets, a similar class of sites was present but with a greater binding capacity (apparent kd approximately 0.56 microgram/mL, R approximately 0.44 microgram/10(9) cells). Gel-filtered platelets that had been stimulated with thrombin displayed two classes of binding sites: a high-affinity class (apparent kd1 approximately 1.1 microgram/mL, R1 approximately 0.39 microgram/10(9) cells) corresponding to that of the unstimulated cells, and a low-affinity class (apparent kd2 approximately 13 micrograms/mL, R2 approximately 2.2 micrograms/10(9) cells). Heparin binding was also increased in platelet-rich plasma when the cells had been stimulated by adenosine diphosphate (ADP) to release, but not when ADP caused primary aggregation without release. Binding was not dependent on extracellular calcium, nor was it reduced by monoclonal antibodies to platelet membrane glycoproteins Ia/IIa, Ib, IIb/IIIa, or IV. Because the apparent dissociation constant of the high-affinity sites (approximately 0.55 microgram/mL) falls in the range of heparin concentrations achieved clinically, these binding sites may be involved in the platelet dysfunction and immune-mediated thrombocytopenia associated with therapeutic heparin. The low-affinity, high-capacity class of sites, which appears after cell stimulation, may participate in the process of platelet adhesion.

Intracellular lactate dehydrogenase concentration as an index of cytotoxicity in rat hepatocyte primary culture.

In searching for a reliable index for cytotoxicity testing in rat hepatocyte primary culture, lactate dehydrogenase (LDH) concentrations in lysates of attached hepatocytes and LDH released into the culture medium were compared under conditions of exposure to various dosages of sodium chloride, sodium salicylate, R-warfarin, acetaminophen, phenylbutazone, and furosemide (frusemide). The amount of intracellular LDH was assessed by inducing the cells to release the enzyme with 0.1% Triton X-100. The induced LDH leakage was completed in 1 hr and the LDH activity was stable in storage at 10 degrees for 2 weeks. We found that intracellular LDH is a direct indicator of the number of viable hepatocytes in contrast to the LDH released, because released LDH does not account for the significant number of cells detached from monolayer but which are not leaky, during the 6-hr test period. Based on IC50 values (50% inhibitory concentration), the relative cytotoxicities are R-warfarin greater than phenylbutazone greater than furosemide greater than acetaminophen greater than sodium salicylate greater than sodium chloride.

Exposure of DNA to methyl mercury results in an increase in the rate of its transcription by RNA polymerase II.

Double-stranded DNA which was exposed to methyl mercury at concentrations of 1 mM and above, and purified by ethanol precipitation and dialysis, was transcribed at a higher rate by RNA polymerase II than was control DNA. The rate of transcription of single-stranded DNA was not affected by similar exposure to methyl mercury. The higher rate of transcription of methyl mercury-treated double-stranded DNA appears to result from a decreased Km of the enzyme for this DNA. This does not appear to result from extensive denaturation, nor from formation of a large number of single-stranded breaks in the DNA.

A comparative study of the effects of mercury compounds on cell viability and nucleic acid synthesis in HeLa cells.

The effects of various mercury compounds on HeLa cell viability and DNA and RNA syntheses in intact cells and in isolated nuclei have been studied. The compounds examined were: methylmercuric chloride, ethylmercuric chloride, dimethylmercury, phenylmercuric acetate, p-hydroxymercuribenzoate, p-hydroxymercuribenzenesulfonate, HgCl2, HgSO4 , Hg(ClO4)2 and Hg2(ClO4)2. All of the compounds except dimethylmercury inhibited colony formation as well as DNA synthesis in intact cells and in isolated nuclei. RNA synthesis in intact cells was inhibited by all the compounds except dimethylmercury, p-hydroxymercuribenzoate and Hg(ClO4)2. In isolated nuclei, alpha-amanitin-resistant RNA synthesis was inhibited by all the compounds except dimethylmercury, alpha-Amanitin-sensitive RNA synthesis was stimulated by some compounds, inhibited by some, and unaffected by others. The effects of two non-mercurial sulfhydryl reagents, N-ethylmaleimide and iodoacetic acid, were also examined. These compounds showed a pattern of effects on nucleic acid synthesis which differed considerably from that of the mercury compounds. Neither compound significantly inhibited alpha-amanitin-resistant RNA synthesis in isolated nuclei, although both inhibited RNA synthesis in intact cells. Iodoacetic acid had no inhibitory effect on DNA synthesis in isolated nuclei but strongly inhibited DNA synthesis in intact cells.

Studies on the mechanism of the stimulation of polymerase II-catalyzed RNA synthesis by mercury compounds.

The specific stimulation of alpha-amanitin-sensitive RNA synthesis in isolated nuclei by methyl mercury (Frenkel, G. D., and Randles, K. (1982) J. Biol. Chem. 257, 6275-6279) has been further investigated. Using the method of alkaline hydrolysis/uridine analysis to determine the number of RNA chains growing in vitro, it was found that the stimulation could not be accounted for by an increase in the number of growing chains. The stimulatory effect of heparin (Coupar, B. E. H., and Chesterton, C. J. (1977) Eur. J. Biochem. 79, 525-533), was found to be additive with that of methyl mercury at saturating concentrations of the latter. Various detergents were found to affect RNA synthesis per se and to modify the stimulatory effect of methyl mercury, suggesting that the stimulation by methyl mercury requires a degree of structural integrity of some nuclear components. The ability of a number of other mercury compounds to stimulate RNA synthesis was investigated. None of the inorganic compounds examined, i.e. HgCl2, HgSO4, and Hg(ClO4)2, stimulated synthesis. Among the alkyl organic compounds tested in addition to methyl mercury, ethyl mercury also stimulated RNA synthesis, but dimethylmercury did not. Among the aryl compounds tested, phenylmercury did not stimulate synthesis whereas p-hydroxymercuribenzoate and p-hydroxymercuribenzenesulfonate did. N-Ethylmaleimide, a nonmercurous sulfhydryl reagent, was found to have only weak ability to stimulate RNA synthesis, compared to a comparable mercury-containing sulfhydryl reagent, p-hydroxymercuribenzoate. The stimulatory effect of the latter was, however, effectively competed out by the former, indicating that sulfhydryl binding is necessary for the stimulation but not sufficient. This conclusion was reinforced by experiments which utilized a model system to measure the ability of various mercury compounds to compete with N-ethylmaleimide in binding to cysteine. The results showed that even compounds such as phenylmercury and the inorganic mercurials, which are unable to stimulate RNA synthesis, are able to bind to a sulfhydryl group.