Increased appearance rate of 27-hydroxycholesterol in vivo in hypercholesterolemia: a possible compensatory mechanism.

BACKGROUND AND AIMS: The first step in the alternative pathway of bile acid biosynthesis is the 27-hydroxylation of cholesterol, which takes place both in liver and extrahepatic tissues. This pathway is believed to play a role in peripheral cholesterol degradation. Aim of this study was to investigate the impact of hyperlipidemia on 27-hydroxycholesterol appearance rate, and to assess the effects induced by treatment with statins. METHODS AND RESULTS: Seven patients with familial hypercholesterolemia and eight patients with familial combined hyperlipidemia underwent determination of 27-hydroxylation rates in vivo by i.v. infusion of deuterated 27-hydroxycholesterol. Isotope enrichment was assayed by gas chromatography-mass spectrometry, allowing to calculate 27-hydroxycholesterol appearance rates. Six normocholesterolemic subjects were regarded as controls. In some hypercholesterolemic patients the infusions were repeated during treatment with atorvastatin or rosuvastatin. Hydroxylation rates were higher in hypercholesterolemic patients (8.7 ± 2.5 mg/h; controls, 3.4 ± 2.0 mg/h; combined hyperlipidemia, 4.4 ± 1.6 mg/h; mean ± SD, P < 0.01 vs both). After statin treatment, both plasma cholesterol levels and hydroxylation rates dropped by nearly 50%. No difference was detectable between the two statins. A linear correlation was shown between plasma cholesterol and 27-hydroxylation rates. CONCLUSION: Hypercholesterolemia associates with increased 27-hydroxycholesterol appearance rates, which decrease during hypocholesterolemic treatment. The correlation with cholesterol levels supports the view that 27-hydroxylation may act as a compensatory mechanism in a condition of larger plasma cholesterol pool. A regulatory role for hepatic and extrahepatic nuclear receptors seems reasonable. These data prompt novel pharmacological approaches for the management of hypercholesterolemia and the prevention of atherosclerosis.

Improved synthesis and radiolabeling of [11C]MP4A, a suitable ligand for the investigation of the cholinergic system using PET.

An improved synthesis of the precursor acetic acid-piperidine-4-yl ester by acetylation of 4-hydroxypiperidine hydrochloride in anhydrous chloroform was developed. A procedure for fast evaluation and characterization of products originated by acetylation of the 4-piperidinol using LC-APCI/MS with an acetonitrile-water gradient method on a Merck Purosphere RP-18 column was also developed. The highly purified precursor allowed the production of [11C]MP4A for PET studies of acetylcholine neurotransmission system. The tracer was produced with >98% radiochemical purity, with yields ranging 20-60% (decay-corrected) from EOB.

Modulation of HMG-CoA reductase activity by pantetheine/pantethine.

The ability of pantetheine/pantethine to modulate the activity of HMG-CoA reductase (EC 1.1.1.34) was determined in vitro with rat liver microsomes. The decay of the activity was obtained with pantethine in the 10(-5)-10(-4) M range, whereas stimulation by pantetheine occurred at 10(-3)-10(-2) M, as previously reported for GSSG and GSH, respectively. Inhibition of HMG-CoA by pantethine in isolated liver cells was also investigated by measuring the enzyme activity in microsomes isolated from hepatocytes incubated without or with 1 mM pantethine under conditions previously shown by us to induce inhibition of cholesterol synthesis from acetate. The enzyme amount was not modified by pantethine, but in cells treated with the disulphide, the relative amounts of the thiolic active forms of the enzyme, both phosphorylated and dephosphorylated, were decreased to about half compared to controls.

Long-term oral L-arginine administration improves peripheral and hepatic insulin sensitivity in type 2 diabetic patients.

UNLABELLED: The aim of this study was to evaluate whether long-term administration of arginine acting through a normalization of NO/cyclic-guanosine-3' 5'-cyclic monophosphate (cGMP) pathway was able to ameliorate peripheral and hepatic insulin sensitivity in 12 lean type 2 diabetic patients. RESEARCH DESIGN AND METHODS: A double-blind study was performed for 3 months. In the first month, patients were treated with their usual diet. Then they were randomly allocated into to groups. In group 1, patients were treated with diet plus placebo (orally three times per day) for 2 months. In group 2 patients were treated for 1 month with diet plus placebo orally, three times per day) and then for 1 month with diet plus L-arginine (3 g three times per day). At the end of the first and the second month of therapy, patients underwent a euglycemic-hyperinsulinemic clamp combined with [6,6-2H2] glucose infusion. A total of 10 normal subjects underwent the same test as control subjects. RESULTS: In group 1, no changes in basal cGMP levels, systolic blood pressure, forearm blood flow, glucose disposal, and endogenous glucose production were observed throughout. In group 2, L-arginine normalized basal cGMP levels and significantly increased forearm blood flow by 36% and glucose disposal during the clamp by 34% whereas it decreased systolic blood pressure and endogenous glucose production by 14 and 29%, respectively. However, compared with normal subjects, L-arginine treatment was not able to completely overcome the defect in glucose disposal. CONCLUSIONS: L-Arginine treatment significantly improves but does not completely normalizc peripheral and hepatic insulin sensitivity in type 2 diabetic patients.

The continuous low dose insulin and glucose infusion test: a simplified and accurate method for the evaluation of insulin sensitivity and insulin secretion in population studies.

In this study we investigated a simple nonlabor-intensive method to evaluate insulin sensitivity and beta-cell function which is suitable for application in population studies. The method is a refinement of the modified Harano test and consists of a continuous low dose insulin (25 mU/kg.h) and glucose (4 mg/kg.min) infusion test (LDIGIT) lasting 150 min. Insulin sensitivity was evaluated as the MCR of glucose divided by the steady state serum insulin level achieved at the end of the test. Insulin secretion was expressed as the incremental area for C-peptide concentration during the first 15 min of the test. We compared the indices of insulin sensitivity and insulin secretion yielded by LDIGIT with those derived from the euglycemic clamp and the hyperglycemic clamp, respectively. Fifty-four subjects underwent a LDIGIT (33 with normal glucose tolerance and 21 with impaired glucose tolerance); of the 54, 19 were submitted to a euglycemic clamp, 18 to a hyperglycemic clamp, and 10 to a modified Harano test (insulin infusion, 50 mU/kg.h; glucose infusion, 6 mg/kg.min). LDIGIT overcame the drawbacks associated with the modified Harano test because it resulted in more stable final glucose levels and prevented the occurrence of hypoglycemic episodes. No significant differences were found between the insulin sensitivity index (ISI) of the LDIGIT and that of the euglycemic clamp for each group of subjects. Moreover, there was a strong correlation between the ISI determined by LDIGIT and the ISI determined by clamp (r = 0.90; P < 0.0001), and the best regression line was not different from the identity line, suggesting that the two indices are equivalent. The index of insulin secretion provided by LDIGIT correlated well with that of the hyperglycemic clamp (r = 0.82; P < 0.001) and was significantly higher in overweight subjects than in normal weight subjects. In conclusion, LDIGIT is a simple and accurate method to assess insulin sensitivity and secretion. It can be useful in population studies and in situations when more complex techniques are not feasible.

Mediation of the hepatic effects of growth hormone by its lipolytic activity.

The aim of the study was to investigate the acute effect of GH per se, independent from its lipolytic activity, on glucose and lipid oxidation and glucose turnover in seven healthy subjects. Five tests lasting 360 min were performed. Each test consisted of a 4-h equilibration period followed by a euglycemic hyperinsulinemic (25 mU/kg x h) clamp lasting 2 h. In test 1 (control experiment) saline was infused, leaving GH and FFA at basal levels. In tests 2, 3, and 4, GH was infused (80 ng/kg x min) to increase GH levels. Whereas in test 2 FFA levels were free to increase due to GH lipolytic activity, in test 3 FFA elevation was prevented by using an antilipolytic compound (Acipimox) that allowed evaluation of the effect of GH at low FFA levels. In test 4 (GH+Acipimox+heparin) GH infusion was associated with the administration of Acipimox and heparin to maintain FFA at the basal level to evaluate the effect of GH per se independent from GH lipolytic activity. In test 5 Acipimox and a variable heparin infusion were given to evaluate possible effects of Acipimox other than the inhibition of lipolysis. During the euglycemic hyperinsulinemic clamp in the presence of high GH and FFA levels (test 2), glucose oxidation was significantly lower and lipid oxidation was significantly higher than in tests 1, 3, 4, and 5. During the same period, hepatic glucose production was completely suppressed in the control study (test 1; 94%) and in test 5 (99.6%), whereas it was significantly less inhibited (65%, 74%, and 73%) when GH was administered in tests 2, 3, and 4. In conclusion, these results suggest that GH directly mediates the reduction of insulin's effect on the liver. In addition, the effect of GH on glucose and lipid oxidation is not direct, but is mediated by its lipolytic activity.

Disposition of metformin (N,N-dimethylbiguanide) in man.

Kinetic parameters of metformin (N,N-dimethylbiguanide), an anti-diabetic reported to be associated with a lower number of episodes of lactic acidosis than phenformin, were determined in volunteers with normal renal function and in patients with different degrees of renal impairment. Drug in body fluids was measured by a highly specific and sensitive mass fragmentographic method, after the formation of a triazine derivative, obtained with heptafluorobutyric anhydride. The half-life (t 1/2) for the elimination of drug from plasma after intravenous injection in 5 normal subjects (1.52 +/- 0.3 hr) (mean +/- SD) was shorter than that reported for phenformin by a similar assay method (7 to 15 hr). The mean t 1/2 in 5 renal patients was 4.94 +/- 1.11 hr, and a correlation was observed between t 1/2 of drug from plasma and creatinine clearance. After oral administration of metformin tablets, drug recovery in urines was only 37.6%, possibly not as a consequence of low bioavailability (a similar low recovery was found after oral administration of the metformin solution used for the intravenous studies), but of binding to the intestinal wall, as shown in animal and clinical studies with metformin and other biguanides. Metformin is rapidly eliminated through active secretion by the kidney (mean renal clearance, 440.8 ml/min)--it is neither metabolized nor protein bound in plasma. The very brief plasma t 1/2 makes significant cumulation, with a standard tid regimen, unlikely. These findings may help explain the lower incidence of toxic effects, particularly lactic acidosis, than after phenformin.

Lack of conversion of xanthine dehydrogenase to xanthine oxidase during warm renal ischemia.

Irreversible transformation of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) during ischemia was determined measuring XDH and total enzyme activity in kidneys before and after 60 min of clamp of the renal pedicle. Tissue levels of adenine nucleotides, xanthine and hypoxanthine were used as indicators of ischemia. After 60 min of clamping, ATP levels decreased by 72% with respect to controls whereas xanthine and hypoxanthine progressively reached tissue concentrations of 732 +/- 49 and 979 +/- 15 nmol.g tissue-1, respectively. Both total and XDH activities in ischemic kidneys (30 +/- 15 and 19 +/- 1 nmol.min-1.g tissue-1) were significantly lower than in controls when expressed on a tissue weight basis. The fraction of enzyme in the XDH form was however unchanged indicating that the reduction of the nucleotide pool is not accompanied by induction of the type-O activity of xanthine oxidase.

Evaluation of three quinoline-carboxamide derivatives as potential radioligands for the in vivo pet imaging of neurodegeneration.

The peripheral-type benzodiazepine receptors (PBRs) are only minimally expressed in normal brain parenchyma, where they are primarily localized in glial cells. Their basal expression rises in different neurodegenerative disorders, due to the presence of infiltrating inflammatory cells and activated microglia. [11C]PK11195, a selective PBR antagonist, has been used for the in vivo PET monitoring of neurodegeneration in clinical observations. We recently developed and labeled with carbon-11 three new carboxamide derivatives: [11C]VC193M, [11C]VC195 and [11C]VC198M. Aim of this study was to evaluate these ligands for the in vivo measuring of PBRs expression in neurodegenerations and compare their kinetic behavior with that of the reference tracer [11C]PK11195. Radioligands were evaluated in a preclinical model of Huntington's disease consisting in the monolateral striatal injection of quinolinic acid (QA). Activated microglia and astrocytic gliosis was present only within the affected striatum. A concomitant increase in radioactivity accumulation was observed for all the tracers examined (P<0.01). Among the new compounds, [11C]VC195 showed higher levels of lesioned/unlesioned striatum ratios (3.28+/-0.44), in comparison with [11C]VC193M and [11C]VC198M (2.69+/-0.53 and 1.52+/-0.36, respectively), but slightly inferior to that observed for [11C]PK11195 (3.76+/-1.41).In conclusion, the results of the study indicate that [11C]VC195 is a promising candidate for in vivo PET monitoring of neurodegenerative processes but its in vivo behavior overlap that of [11C]PK11195.

Hypocaloric high-protein diet improves glucose oxidation and spares lean body mass: comparison to hypocaloric high-carbohydrate diet.

The aim of the study was to investigate the effects of two hypocaloric (800-kcal) diets on body weight reduction and composition, insulin sensitivity, and proteolysis in 25 normal glucose-tolerant obese women. The two diets had the following composition: 45% protein, 35% carbohydrate (CHO), and 20% fat (HP diet, 10 subjects), and 60% CHO, 20% protein, and 20% fat (HC diet, 15 subjects); both lasted 21 days. A euglycemic hyperinsulinemic (25 mU/kg/h) clamp lasting 150 minutes combined with indirect calorimetry was performed before and after the diet. Both diets induced a similar decrease in body weight and fat mass (FM), whereas fat-free mass (FFM) decreased only after the HC diet. 3-Methylhistidine (3-CH3-HIS) excretion was reduced by 48% after the HP diet and remained unchanged after the HC diet (P < .05). A significant correlation was found between the changes in FFM and in 3-CH3-HIS excretion after the diet (rs = .50, P < .02). Blood glucose remained unchanged, while insulin decreased in both diets. Free fatty acids (FFA) significantly increased only after the HC diet (P < .05). During the clamp period, glucose disposal and glucose oxidation significantly increased after the HP diet and significantly decreased after the HC diet. Opposite results were found when measuring lipid oxidation. In conclusion, our experience suggests that (1) a hypocaloric diet providing a high percentage of natural protein can improve insulin sensitivity; and (2) conversely, a hypocaloric high-polysaccharide-CHO diet decreases insulin sensitivity and is unable to spare muscle tissue.

Glucose turnover and insulin clearance after growth hormone treatment in girls with Turner's syndrome.

The study was performed to elucidate, by means of a euglycemic-hyperinsulinemic clamp, whether insulin sensitivity, lipid levels, posthepatic insulin delivery, and insulin clearance are impaired in girls with Turner's syndrome in the absence of previous treatment (T0) and after 6 (T6) and 12 (T12) months of growth hormone (GH) therapy (GHT). The study was performed in six girls with Turner's syndrome and eight healthy girls. We found that previously untreated girls with Turner's syndrome had a normal insulin activity on glucose metabolism. GHT progressively and significantly decreased hepatic insulin sensitivity. In fact, residual hepatic glucose release (HGR), which was 19.6 +/- 4.7 mg/m2. min at T0, doubled at T6 (39.3 +/- 5.1 mg/m2.min) and showed a threefold increase at T12 (68.7 +/- 10.8 mg/m2.min, P < .05 v T0). On the contrary, GHT did not show an appreciable influence on peripheral insulin sensitivity. Insulin clearance was higher in girls with Turner's syndrome than in control girls at T0 (30.0 +/- 2.8 v 20.2 +/- 1.1 mL.kg-1.min-1). It decreased to normal values at T6 (18.2 +/- 2.0 mL.kg-1.min-1, P < .05 v T0) and remained at normal levels at T12 (23.8 +/- 2.9 mL.kg-1. min-1). The posthepatic insulin delivery rate significantly increased at T6 and T12, suggesting increased insulin secretion. In conclusion, we found that insulin-stimulated glucose turnover was normal in girls with Turner's syndrome before therapy. One year of GHT was successful in stimulating the growth rate, but significantly decreased the insulin suppressibility on HGR with only slight changes in peripheral insulin sensitivity. In addition, an increase in the insulin posthepatic delivery rate and a normalization of insulin clearance were present, probably to counteract hepatic insulin resistance.

Hydrolysis of cyclosporin A: identification of 1,11 seco-cyclosporin A and 4,5 seco-isocyclosporin A by FAB-MS/MS.

We have previously reported that treatment of CsA with aqueous HCI gives rise to the formation of a number of water-soluble compounds. Two of these were identified from their FAB-MS/MS spectra as open-chain nona- and decapeptides. We describe here the identification of two other main compounds deriving from the same treatment. Identification was rendered possible from the comparison of their FAB-MS/MS spectra with those of methyl and acetyl derivatives. The two compounds are water-soluble, open-chain undecapeptides corresponding to 1.11 seco-CsA and of 4.5 seco-isoCsA, respectively.

Quantitation of cyclosporin A in whole blood by liquid chromatography/stable isotope dilution electrospray ionization mass spectrometry.

Therapy with cyclosporin A (CsA) for immunosuppression after organ transplantation requires monitoring of its levels in blood owing to the narrow therapeutic index of the drug and to the high inter-individual variability of the drug absorption and metabolism. We describe the preparation of CsA labelled with stable isotopes ((13)C and (2)H) with an isotopic enrichment of about 99% using labelled glucose and its use as internal standard for quantification of CsA blood levels by isotope dilution/electrospray ionization mass spectrometry. The method was found to be linear in the tested range (1-1000 ng) with and without the matrix. The accuracy of the bracketting calibration curves prepared using 100 ng ml(-1) labelled CsA was within +/-1.7% (bias). The results confirmed the usefulness of the procedure as a reference method for the external quality assessment of the field methods for the evaluation of CsA blood concentration, the imprecision (relative standard deviation) and accuracy (bias) being <2%.

Synthesis and in vivo evaluation of [(11)C]CGP62349, a new GABA(B) receptor antagonist.

This paper describes the radiosynthesis of [(11)C]CGP62349, a potential ligand to assess GABA(B) receptors in vivo. (11)C was introduced by O-methylation of the corresponding des-methyl precursor, namely CGP67780. The final product was obtained with a reliable method in good yield. The radioligand was tested in monkey, revealing negligible blood-brain barrier penetration and brain uptake, thus prompting us to search for a new target structure with a better lipophilicity.

The effect of cholestyramine on liver HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in various laboratory animals.

The activity of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase was assayed in the liver of rats, rabbits, hamsters and guinea pigs at the minimum of the day cycle and after one night fasting. The amount of HMG-CoA reductase, as determined after its complete dephosphorylation in vitro was of the same order of magnitude in the tested species. The dephosphorylated active form of the enzyme was detectable only in the rat. Cholesterol 7 alpha-hydroxylase activity was also much higher in the rat. Cholestyramine treatment stimulated the activity of both enzymes. In particular, the ratio between active and inactive HMG-CoA reductase in rabbits, hamsters and guinea pigs became of the same order of magnitude of that found in rats.

HMGCoA reductase and cholesterol 7 alpha-hydroxylase in human liver.

The activities of HMGCoA reductase and cholesterol 7 alpha-hydroxylase were assayed in liver biopsies of patients with or without cholestyramine treatment. The active dephosphorylated form of HMGCoA reductase and the activity of cholesterol 7 alpha-hydroxylase were under the detection limits in untreated subjects. After cholestyramine treatment activities of both enzymes were stimulated and the active form of HMGCoA reductase became detectable in four out of the five tested patients. In two subjects who received cholestyramine, the effect of exogenously added [4-14C] cholesterol on cholesterol 7 alpha-hydroxylase was tested. In the presence of Tween 80, the detergent by which [14C]cholesterol was suspended, the enzyme activity was profoundly inhibited and synthesis of 7 alpha-hydroxycholesterol was extremely low.

Diabetes-induced alteration of HMGCoA reductase forms in rat livers.

The effects of streptozotocin-induced diabetes on the various forms of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA reductase), phosphorylated/dephosphorylated and thiolic/disulphide, were studied in rat liver. Animals were treated twice with 65 mg/kg intraperitoneally of streptozotocin to induce diabetes and sacrificed after 5 days. The relative amounts of the four possible forms of the enzyme were determined in control and diabetic rats. As determined from the total activity, and in agreement with previous reports, the enzyme protein was significantly decreased in streptozotocin-treated rats. However, the percentage of the active thiolic dephosphorylated form was higher in these animals than in controls, suggesting a response of the liver to the decrease both total and specific activity of HMGCoA reductase.

Inhibition of cholesterol synthesis and hepatic 3-hydroxy-3-methylglutaryl--CoA reductase in rats by simvastatin and pravastatin.

In this communication we attempt to provide one possible explanation for the observed differences regarding kinetics and distribution between simvastatin and pravastatin. Rats treated with simvastatin or pravastatin exhibited a reduction in the incorporation of [2-(14)C] acetate into liver cholesterol and displayed lower plasma mevalonate levels as compared to control animals. Moreover, both the total and dephosphorylated 3-hydroxy-3-methylglutaryl--CoA (HMG-CoA) reductase (EC 1.1.1.34) activities, particularly 1 h after treatment, were greatly reduced in liver microsomes obtained from simvastatin-treated as compared to control rats. During the same time frame, these parameters were actually elevated with pravastatin treatment. It is known that HMG-CoA reductase synthesis and activity increase following their competitive inhibition. Our results suggest that pravastatin, at 1 h following treatment, was no longer bound to the enzyme; however, it had entered the liver because its inhibitory effect on cholesterol synthesis was manifest at early times after administration. These data provide a plausible rationale for the earlier observation that activity of simvastatin persists longer in plasma than does that of pravastatin.

Synthesis of [O-methyl-11C]fluvoxamine--a potential serotonin uptake site radioligand.

5-Methoxy-1-[4-(trifluoromethyl)-phenyl]-1-pentanone-O-(2-aminoethyl)oxi me (fluvoxamine), a potent clinically used antidepressant, was labelled with carbon-11 (t1/2 = 20.4 min) as a potential radioligand for the non-invasive assessment of serotonin uptake sites in the human brain with positron emission tomography (PET). The two-step radiochemical synthesis consisted of O-methylation of an amino-protected desmethyl precursor with [11C]methyl iodide under mild conditions in the presence of tetrabutylammonium hydroxide in acetonitrile, followed by deprotection with trifluoroacetic acid. 5-[11C]Methoxy-1-[4-(trifluoromethyl)-phenyl]-1-pentanone-O-(2-aminoethy l) oxime was obtained in > 98% radiochemical purity in 40 min with a radiochemical yield of 4 +/- 2% (non-decay corrected) and a specific radioactivity of 1 +/- 0.5 Ci/mumol. 5-Hydroxy-1-[4-(trifluoromethyl)-phenyl]-1-pentanone-O-[2- (tert-butoxycarbonylamino)ethyl]oxime, the precursor for the radiosynthesis of [11C]fluvoxamine, was prepared by a convenient three-step synthesis from the pharmaceutical form of fluvoxamine maleate by converting it into the free base, demethylation by trimethyliodosilane and introduction of the BOC-protective group with di-tert-butyl dicarbonate.

Radiosynthesis of [123I]betaCIT, a selective ligand for the study of the dopaminergic and serotoninergic systems in human brain.

The procedure previously reported for the radiosynthesis of [123I]betaCIT was modified in order to improve both radiochemical yield and purity of betaCIT (2beta-carbomethoxy-3beta(4-iodophenyl) tropane) to be injected for SPECT (Single Photon Emission Computed Tomography) analysis imaging. The overall procedure, involving a HPLC purification step, results in quite good and reproducible yields of a highly purified tracer.