Evidence that the major oxysterols in human circulation originate from distinct pools of cholesterol: a stable isotope study.

The major oxysterols in human circulation are 7 alpha-, 27-, and (24S)-hydroxycholesterol. Two unique experiments were performed to elucidate their origin and kinetics. A volunteer was exposed to (18)O(2)-enriched air. A rapid incorporation of (18)O in both 7 alpha- and 27-hydroxycholesterol and disappearance of label after exposure were observed. The half-life was estimated to be less than 1 h. Incorporation of (18)O in (24S)-hydroxycholesterol was not significant. In the second experiment a volunteer was infused with liposomes containing 10 g of [(2)H(6)]cholesterol. This resulted in an enrichment of plasma cholesterol with (2)H of up to 13%, and less than 0.5% in cerebrospinal fluid cholesterol. The content of (2)H in circulating 7 alpha-hydroxycholesterol remained approximately equal to that of plasma cholesterol and decreased with a half-life of about 13 days. The (2)H content of circulating 27-hydroxycholesterol was initially lower than that of cholesterol but in the last phase of the experiment it exceeded that of cholesterol. No significant incorporation of (2)H in (24S)-hydroxycholesterol was observed. It is evident that 7 alpha-hydroxycholesterol must originate from a rapidly miscible pool, about 80% of 27-hydroxycholesterol from a more slowly exchangeable pool, and more than 90% of (24S)-hydroxycholesterol from a nonexchangeable pool, presumably the brain. The results are discussed in relation to the role of oxysterols in cholesterol homeostasis and their use as markers for pathological conditions. - Meaney, S., M. Hassan, A. Sakinis, D. Lütjohann, K. von Bergmann, A. Wennmalm, U. Diczfalusy, and I. Björkhem. Evidence that the major oxysterols in human circulation originate from distinct pools of cholesterol: a stable isotope study. J. Lipid Res. 2001. 42: 70;-78.

An 18oxygen inhalation method for determination of total body formation of nitric oxide in humans.

The formation of nitric oxide (NO) and the subsequent conversion of the NO formed into nitrate require molecular oxygen. Based on this fact, we have recently developed a method using inhalation of the stable oxygen isotope, i.e. 18O2, to determine total formation of NO in small laboratory animals. The method has now been further developed to be applicable also in humans. Five healthy awake male subjects inhaled a gas mixture of unlabelled and 18-labelled oxygen (approximate ratio 4:1) in nitrogen from a closed breathing system equipped with eliminators for carbon dioxide and water vapour. The ratio of unlabelled to 18-labelled oxygen, as well as the total oxygen concentration during the inhalation, were monitored. Venous blood samples were taken before and after the inhalation for analysis of unlabelled and 18O-labelled nitrate by gas chromatography/mass spectrometry. The procedure was repeated with the same protocol on a later occasion, during ongoing treatment with the NO synthesis inhibitor NG-monomethyl-L-arginine (L-NMMA). The average nitrate level in plasma in the absence of L-NMMA was 26 micromol l-1. The rate of total synthesis of NO was estimated to be 0.38 +/- 0.06 mu mol kg-1 h-1, corresponding to a total body formation of 600-700 mu mol/24 h in an adult male. Infusion of L-NMMA caused an increase in mean arterial blood pressure from 86 +/- 4 to 99 +/- 5 mmHg (P<0.05). The average plasma level of nitrate during infusion of L-NMMA was 24 mu mol l-1. NO formation during infusion of L-NMMA was 0.17 +/- 0.03 mu mol kg-1 h-1, i.e. significantly (P<0.05) lower than in the absence of L-NMMA. We suggest that the described method allows direct determination of total NO formation in man. The method may be useful in the study of various experimental and pathophysiological conditions affecting NO formation.

Estimation of total rate of formation of nitric oxide in the rat.

Nitric oxide (NO) is a powerful mediator with important actions in several organ systems. NO is synthesized during the enzymatic conversion of l-arginine and molecular oxygen to L-citrulline. About 90% of the NO formed is degraded to nitrate. Utilizing this information we have developed a method for assessment of the total rate of formation of NO in the rat. Male Wistar rats were kept in a closed-cage system allowing controlled breathing of a mixture of 18O2 and 16O2 in N2 for up to 5h. Blood samples for mass spectrometric analysis of nitrate residues with varying numbers of 18O atoms incorporated were drawn before and during the exposure to 18O2. By comparing the relative incorporation of 18O into nitrate residues to the 16O2/18O2 ratio in the breathing gas mixture in the cage system it was possible to calculate the absolute rate of NO formation in the animal. The rate of formation of NO in anaesthetized rats ranged from 0.33 to 0.85 micromol.kg-1.h-1. The rate of formation did not differ significantly in rats which were awake during the experiment (range 0.36-0.72 micromol.kg-1.h-1). The L-arginine analogue Nomega-nitro-L-arginine methyl ester (L-NAME) dose-dependently inhibited the formation of NO, at a dose of 100mg/kg by more than 99%. The technique presented allows estimation of the total rate of formation of NO in vivo in rats. Application of the technique may yield important information about the physiological and pathophysiological roles of NO. It may also be utilized to evaluate the effect of pharmacological treatment on NO formation.

Importance of a novel oxidative mechanism for elimination of brain cholesterol. Turnover of cholesterol and 24(S)-hydroxycholesterol in rat brain as measured with 18O2 techniques in vivo and in vitro.

The brain is the most cholesterol-rich organ in the body. Brain cholesterol is characterized by a very low turnover with very little exchange with lipoproteins in the circulation. Very recently we showed that there is a continuous age-dependent flux of 24(S)-hydroxycholesterol from the human brain into the circulation (Lütjohann, D., Breuer, O., Ahlborg, G., Nennesmo, I., Sidén, A., Diczfalusy, U., and Björkhem, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9799-9804). Here we measured the rate of synthesis of cholesterol as well as the conversion of cholesterol into 24(S)-hydroxycholesterol in rat brain in vivo with use of an 18O2 inhalation technique and mass isotopomer distribution analysis. Cholesterol synthesis was found to correspond to 0.03 +/- 0.01% of the pool per h. Conversion of cholesterol into 24(S)-hydroxycholesterol was of a similar magnitude, about 0.02% of the pool per h. Brain microsomes converted endogenous cholesterol into 24(S)-hydroxycholesterol at a similar rate when incubated in the presence of NADPH. When incubated with whole homogenate and subcellular fractions of rat brain, there was no significant conversion of tritium-labeled 24-hydroxycholesterol into more polar products. Plasma from 18O2-exposed rats contained 24(S)-hydroxycholesterol with an enrichment of 18O similar to that in 24(S)-hydroxycholesterol in the brain. The results suggest that the present 24(S)-hydroxylase mediated mechanism is most important for elimination of cholesterol from the brain of rats. There is a slow conversion of brain cholesterol into 24(S)-hydroxycholesterol with a rapid turnover of the small pool of the latter oxysterol due to leakage to the circulation (half-life of brain 24(S)-hydroxycholesterol is about 0.5 days as compared with 2-4 months for brain cholesterol). It is evident that the 24(S)-hydroxylation greatly facilitates transfer of cholesterol over the blood-brain barrier and that this hydroxylation may be critical for cholesterol homeostasis in the brain.

Transformation of subcutaneous nitric oxide into nitrate in the rat.

Following its addition to arterialized blood in vitro, nitric oxide (NO) is transformed into nitrate in the erythrocytes. Inhaled NO is similarly transformed into nitrate in the blood in vivo. These observations suggest that nitrate is a universal end-metabolite of NO, i.e. of endogenously formed NO as well. However, endogenous NO may also be inactivated in tissues, i.e. outside the vascular lumen. To study the fate of NO metabolized with delayed access to the blood, rats were given subcutaneous injections of 15NO or K15NO3, and the plasma concentrations of 15NO3(-) were followed for 450 min after injection. The values for the distribution volume and plasma decay (t12) of 15NO3(-) did not differ between rats given 15N-labelled NO and NO3(-). The area under the plasma decay curve for rats given 15NO amounted to 89% of the corresponding area for animals given K15NO3. This demonstrates that 15NO, when given extravascularly in millimolar concentrations, is mainly transformed into 15N-labelled nitrate. Other rats were kept in an atmosphere containing a mixture of 16O2 and 18O2. Nitrate residues containing either one or two 18O atoms were isolated from the blood, indicating that inhaled oxygen was incorporated during both the formation of NO and the subsequent transformation of NO into nitrate. The fraction of nitrate residues containing two 18O atoms was larger than that containing one 18O atom. We propose that nitrate is a major stable metabolite of endogenous NO that does not primarily diffuse into the vascular lumen following formation. Hence nitrate seems to be the quantitatively most important end-product of the metabolism of endogenous NO. The transformation of endogenous NO into nitrate involves the incorporation of inhaled oxygen.

Experimental evaluation of a new self-expanding patent ductus arteriosus occluder in a canine model.

PURPOSE: A new self-expanding patent ductus arteriosus (PDA) occluder was designed. MATERIALS AND METHODS: Percutaneous closure of surgically created aortopulmonary shunts was attempted in 19 dogs. The occlusion device consisted of a nitinol wire frame tightly woven into a cylinder with a flat retention disc. A polyester-filled frame was used in the last six procedures. A 6-F introducing sheath was advanced across the aortopulmonary conduit into the descending thoracic aorta. The prosthesis (attached on a stiff delivery cable) was advanced through the introducing sheath. The retention disc was first released in the descending thoracic aorta, then the cylindrical device frame was expanded within the conduit by withdrawing the sheath. RESULTS: Subtotal misplacement into the descending aorta occurred in one procedure (overall technical success rate, 95%), and one animal died before the 1-week follow-up. Complete angiographic shunt closure was achieved in seven of 18 (39%) animals at 30 minutes, 12 of 17 (71%) animals at 1 week, 14 of 17 (82%) animals at 1 month, and 11 of 12 (92%) animals at 3 months. Significantly higher 30-minute closure rates occurred with polyester-filled occluders compared with nonfilled occluders (five of five [100%] vs one of 13 [15%]; P = .002). Persistent shunt at 3 months occurred in only one nonfilled device (6%). In the remaining 16 animals, both orifices of the shunt were covered by a smooth glistening neoendothelium at postmortem examination. CONCLUSION: This device combines the advantages of small delivery system, easy placement, self-centering, and repositionability. Immediate shunt closure can be reliably accomplished with the polyester-filled prosthesis.

The insulin secretory response to intravenous glucose in the rat is independent of NO formation.

In isolated pancreative beta cells from rats the insulin secretory response to glucose is amplified by L-arginine. Since this effect is inhibited by NO synthesis inhibitors, and since L-arginine is precursor of NO, the observation indicates a role for NO in insulin secretion from beta cells. We recently reported that i.v. L-arginine elicited insulin secretion in anaesthetized rats by a mechanism that was partly NO dependent. The aim of the present study was to assess if the insulin secretory response to an intravenous infusion of glucose also requires an intact NO formation. Anaesthetized rats were given D-glucose (100 mg kg-1 min-1 i.v. for 30 min). Plasma insulin (PI), blood glucose (BG) levels and mean arterial blood pressure (MAP) were assessed from before and until 15 min after the end of the infusion. One group of rats were untreated and served as controls. The two other groups were pretreated with either of the NO synthase inhibitors NW-nitro-L-arginine methyl ester (L-NAME, 50 mg kg-1 i.v.), or NG-monomethyl-L-arginine (L-NMMA, 100 mg kg-1 i.v.). In controls infusion of glucose elevated PI by up to 25 +/- 3 U L-1, and BG by up to 27 +/- 1 mmol L-1. Pretreatment with L-NAME elevated MAP from 74 +/- 6 to 132 +/- 4 mmHg, indicating that NO synthase was inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)