The relative importance of cyclosporine exposure in heart, kidney or liver transplant recipients on maintenance therapy.

We investigated the relationship between cyclosporine exposure and the presence of cyclosporine-related side effects and assessed the advantage of the cyclosporine concentration 2 h post-dose (C(2)) over pre-dose concentration (C(0)) monitoring. Cyclosporine area-under-the-concentration-time curves were measured during the absorption phase (AUC(0-4 h)) in 49 liver, 28 heart and 26 kidney transplant recipients (time since transplantation >6 years) with or without cyclosporine-related side effects on maintenance therapy. The cyclosporine C(0) correlated well with AUC(0-4) (r=0.77), whereas C(2) levels correlated strongly with AUC(0-4) (r=0.92). Although we observed a trend towards higher CsA concentrations in transplant recipients with side effects than in patients without CsA toxicity, the large majority of those differences were not statistically significant. Thus, as cyclosporine exposure was not clearly related to the presence of side effects, and C(0) correlated fairly with AUC(0-4), the advantage of monitoring cyclosporine treatment using C(2) rather than C(0), may be limited for patients on cyclosporine maintenance therapy.

Cyclosporine is associated with endothelial dysfunction but not with platelet activation in renal transplantation.

BACKGROUND: Cyclosporine (CsA) is associated with thrombotic micro-angiopathy and endothelial dysfunction. Markers of endothelial dysfunction may serve to identify patients at risk for development of vascular injury. In this study we measured von Willebrand Factor (vWF) and sP-selectin as possible markers for endothelial dysfunction in renal transplant recipients at different concentrations of CsA. Because sP-selectin can also be derived from platelets an additional in vitro study was performed to study the potential effect of CsA on the expression of P-selectin on platelet surface, while the effects of CsA on the interaction of platelets with Endothelial Cell Matrix (ECM) were studied under flow conditions in a perfusion chamber model. METHODS: CsA was stepwisely replaced by mycophenolate mofetil (MMF) in 15 renal transplant recipients (more than 6 months after transplantation). VWF and sP-selectin were measured at normal CsA (median trough level 130 microg/l), low CsA (trough level 45 microg/l) and after stopping CsA. MMF 2 g daily was added while lowering and stopping CsA. Platelet activation was investigated by measurement of P-selectin on platelet surface by flow-cytometry (FACS), after incubation with CsA (0, 2, 20 and 200 mg/l) in vitro and after perfusion of whole blood over ECM with CsA (0 or 2 mg/l, peak levels). RESULTS: Stepwise withdrawal of CsA gave a dose-related decrease of both vWF and sP-selectin, suggesting reversible endothelial dysfunction. FACS showed no expression of P-selectin on platelets by CsA. Also perfusion studies over ECM demonstrated no platelet activation by CsA but even inhibition of platelet adhesion and aggregation. CONCLUSIONS: The use of CsA is not accompanied by platelet activation. However endothelial dysfunction induced by CsA does occur as reflected by increased vWF and sP-selectin. (See Editorial p. 1).

Correction of ECG variations caused by body position changes and electrode placement during ST-T monitoring.

BACKGROUND: Electrocardiogram variations (ECG) due to body position changes and electrode placements are common problems of continuous ST-T monitoring. Body position changes may cause QRS and ST-T changes and trigger false alarms. Placement of arm and leg electrodes in a coronary care unit environment is usually near the thorax instead of standard position at the wrists and ankles. This may affect the limb leads and complicate diagnostic interpretation. The purpose of this study was to assess the effects of these sources of ECG variation and to correct for them. Continuous 12-lead ECG recordings were obtained from 160 patients admitted to the coronary care unit. Each patient underwent a body position test (supine, left-lateral, and upright position). Scalar and spatial approaches were investigated for reconstruction of the ECG in supine position. The scalar approach uses linear regression. The spatial approach transforms the ECG into a derived vestorcardiogram. The spatial QRS-loop is then rotated and scaled to match the vector loop in supine position and transformed back to a 12-lead ECG. MATERIALS AND METHODS: To assess the effect of electrode placement, monitoring and standard limb leads were simultaneously recorded in a group of 80 patients. To map the monitoring leads to standard leads, general and patient-specific reconstruction coefficients were derived by linear regression from half of the patients and tested on the other half. Similarity between the reference and reconstructed ECGs was measured by correlation, similarity coefficient [(SC=1-RMS(residual error)/RMS(signal)], and difference in frontal QRS-Axis. RESULTS AND CONCLUSION: Only 14% (23 of 160) of the patients showed marked ECG changes (ST elevations, QRS-axis shifts, T-wave inversions). The scalar method (median correlation > 0.994, SC > 0.902, QRS axis difference 0 degrees) performed better than spatial (median correlation 0.946, SC > 0.792, QRS axis difference 0 degrees). Monitoring leads can be mapped to standard limb leads in good to excellent approximaiton. General reconstruction (median correlation 0.993 and SC 0.764) performed slightly worse than patient-specific reconstruction (median correlation 0.997 and SC 0.908).

Nifedipine improves endothelial function in hypercholesterolemia, independently of an effect on blood pressure or plasma lipids.

OBJECTIVE: Dihydropyridine calcium antagonists have been shown to retard atherogenesis in animal models and to prevent the development of early angiographic lesions in human coronary arteries. Endothelial dysfunction is an early event in the pathogenesis of cardiovascular disease. We investigated whether nifedipine could improve endothelial function in hypercholesterolemia, independently of changes in blood pressure or plasma lipids. METHODS: First, we compared in vivo forearm vascular responses to the endothelium-dependent and independent vasodilators serotonin (5-HT) and sodium nitroprusside (SNP) in 11 patients with familial hypercholesterolemia before and after 6-weeks treatment with nifedipine GITS (60 mg, OD) and in 12 matched controls. In a subgroup of six control subjects forearm vascular function was also assessed before and after 6-weeks nifedipine GITS treatment. In vitro, we subsequently explored possible mechanisms underlying the effect of nifedipine on endothelial function. We investigated the effects of nifedipine on both NO production by recombinant endothelial NO synthase (eNOS) and endothelial cells, using 3H-arginine conversion, as well as on superoxide generation by endothelial cell lysates, using lucigenin enhanced chemiluminescence. RESULTS: In hypercholesterolemia 5-HT-induced vasodilation was impaired (47 +/- 9% increase in forearm bloodflow vs. 99 +/- 8% in controls). Treatment with nifedipine completely restored 5-HT-induced vasodilation (113 +/- 13%), whereas it did not influence basal forearm vasomotion or SNP-induced vasodilation. Nifedipine did not alter forearm vascular responses in control subjects and did not alter blood pressure or plasma lipids. In vitro, we found no direct effect of nifedipine on NO production by recombinant eNOS or endothelial cells. However, we did observe a reduction in endothelial superoxide generation. CONCLUSIONS: Our data show that nifedipine improves endothelial function in hypercholesterolemia. It is suggested from our in vitro experiments that this effect is due to reduced NO degradation.

5-methyltetrahydrofolate, the active form of folic acid, restores endothelial function in familial hypercholesterolemia.

BACKGROUND: Impaired nitric oxide (NO) activity is an early event in the pathogenesis of cardiovascular disease, resulting from either reduced NO formation or increased NO degradation. Administration of tetrahydrobiopterin (BH4), an essential cofactor for NO production, could restore NO activity in familial hypercholesterolemia (FH). Because folates have been suggested to stimulate endogenous BH4 regeneration, we hypothesized that administration of 5-methyltetrahydrofolate (5-MTHF, the active circulating form of folate) might improve NO formation in FH. METHODS AND RESULTS: We studied the effects of 5-MTHF on NO bioavailability in vivo in 10 patients with FH and 10 matched control subjects by venous occlusion plethysmography, using serotonin and nitroprusside as endothelium-dependent and -independent vasodilators. In vitro, we investigated the effect of 5-MTHF on NO production by recombinant endothelial NO synthase (eNOS) by use of [3H]arginine to [3H]citrulline conversion. We also studied the effects of 5-MTHF on superoxide generation by eNOS and xanthine oxidase (XO) by use of lucigenin chemiluminescence. The impaired endothelium-dependent vasodilation in FH (63% versus 90% in control subjects) could be reversed by coinfusion of 5-MTHF (117% vasodilation), whereas 5-MTHF had no significant effect on endothelium-dependent vasodilation in control subjects. 5-MTHF did not influence basal forearm vasomotion or endothelium-independent vasodilation. 5-MTHF had no direct effect on in vitro NO production by eNOS. However, we did observe a dose-dependent reduction in both eNOS- and XO-induced superoxide generation. CONCLUSIONS: These results show that the active form of folic acid restores in vivo endothelial function in FH. It is suggested from our in vitro experiments that this effect is due to reduced catabolism of NO.

Tetrahydrobiopterin regulates superoxide and nitric oxide generation by recombinant endothelial nitric oxide synthase.

Nitric oxide (NO) produced by the endothelial isoform of nitric oxide synthase (NOS III) is a key determinant of the anti-atherosclerotic properties of the endothelium. Recent in vivo studies suggest that NOS III may also be a source of superoxide production, which would limit its role as a NO-producing enzyme. In the current study we examined both the NO and the superoxide generating potential of recombinant NOS III obtained from a baculovirus/Sf9 expression system. Using lucigenin chemiluminesence we could indeed demonstrate (superoxide dismutase inhibitable) superoxide production by NOS III. This superoxide production was not affected by administration of L-arginine, but could be inhibited dose-dependently by the co-factor tetrahydrobiopterin (BH4). BH4 also dose dependently decreased superoxide generation by hypoxanthine/xantine oxidase suggesting a direct antioxidant effect. Superoxide generation by NOS III could be completely inhibited by diphenyleneiodonium (DPI), an inhibitor of the flavin moiety of the enzyme, indicating that this group is a main source of superoxide production by the enzyme. Using measurement of [3H-L-arginine] conversion to [3H-L-citrulline], it appeared that BH4 directly increased the production of NO by NOS III. In addition, we observed that BH4 stablized the NOS III in its dimeric form, suggesting that an effect on allosteric conformation could be involved in this effect on NO production. NOS III thus appears to be a superoxide generating enzyme probably through its flavin moiety, as well as a BH4-dependent NO producing enzyme.

Hereditary tyrosinemia type I: lack of correlation between clinical findings and amount of immunoreactive fumarylacetoacetase protein.

Immunoblot analyses with bovine fumarylacetoacetase antibodies have been performed in fibroblast extracts from 28 patients with hereditary tyrosinemia of various clinical phenotypes, in one healthy individual homozygous for a "pseudodeficiency" gene for fumarylacetoacetase, and in three tyrosinemia families in which one or both parents are compound heterozygotes for the tyrosinemia and pseudodeficiency genes. Liver extracts from two chronic patients were also investigated. None of the patients with the acute type of tyrosinemia had detectable immunoreactive protein in fibroblast extracts. Only two of seven patients with typical chronic tyrosinemia had definite immunoreactivity in fibroblasts. In liver tissue, one of the patients had cross-reactive material and the other had no immunoreactivity. Four of 13 patients with intermediate clinical findings showed immunoreactivity in fibroblasts. There was no relationship between severity of symptoms and amount of cross-reactive material in this group. The pseudodeficiency gene product gave almost no detectable immunoreactivity in fibroblasts. The results indicate that chronic tyrosinemia may be due to at least two protein variants, and immunoblotting does not classify tyrosinemia patients according to clinical findings.