Background: Determinations of glucose turnover based on infusion of deuterium-labelled glucose are frequently undertaken in studies involving the pathophysiology of diabetes or obesity or the metabolic response to physical exercise. Methods: A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for determination of the glucose-d(2)/glucose ratio in human serum and plasma following the intra-venous infusion of 6,6-d(2)-glucose. Atmospheric pressure chemical ionization measuring negative ions in selected reaction monitoring mode (product ions at m/z 179 -> 89 and m/z 181 -> 89) was used. The serum or plasma samples (50 mu L) were prepared by protein precipitation with acetonitrile and the resulting supernatant was directly used for the LC-MS/MS analysis. The chromatography was performed on a Luna 3 micron NH2 100A column (100 x 2 mm) using a mobile phase containing 85% acetonitrile with 20 mmol/L of ammonium acetate at a flow rate of 400 mu L/min and an oven temperature of 40 degrees C. Results: A linear response was obtained for the glucose-d(2)/glucose ratio over the relative concentration range of 0-10%, r(2) > 0.999. The peak area ratio was determined with an imprecision of 1.20-8.19% (coefficient of variation). The ion suppression from matrix compared to water was in the order of 55%. Chromatographic retention time was between 4 and 5 min and the total analysis time was 10 min. The validated method was successfully applied for the analysis of human serum samples from a clinical study involving infusion of 6,6-d(2)-glucose and evaluation of endogenous glucose production. Conclusions: It is concluded that the described method provides an easy and precise technique for the determination of serum and plasma glucose-d(2)/glucose ratios in clinical studies.

A new isotope dilution LC-MS/MS method for assay of 7alpha-hydroxy-4-cholesten-3-one without need for derivatization is described. This method was used in catheterization experiments on healthy fasting volunteers. The levels of this generally used marker for bile acid synthesis were slightly but significantly higher in the hepatic vein than in the brachial artery. In contrast, the levels of the precursor to 7alpha-hydroxy-4 cholesten-3-one, 7alpha-hydroxycholesterol, were the same in the two vessels. It is concluded that there is a net extrahepatic metabolism of 7alpha-hydroxy-4-cholesten-3-one. The similarity and very high correlation between the levels in the two vessels (r=0.97) are consistent with the contention that 7alpha-hydroxy-4-cholesten-3-one is a suitable marker for the activity of the hepatic cholesterol 7alpha-hydroxylase and thus bile acid synthesis.

Recently, we demonstrated a net blood-to-brain passage of the oxysterol 27-hydroxycholesterol corresponding to 4-5 mg/day. As the steady-state levels of this sterol are only 1-2 mu g/g brain tissue, we hypothesized that it is metabolized and subsequently eliminated from the brain. To explore this concept, we first measured the capacity of in vitro systems representing the major cell populations found in the brain to metabolize 27-hydroxycholesterol. We show here that 27-hydroxycholesterol is metabolized into the known C-27 steroidal acid 7 alpha-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7 alpha-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid in plasma from the internal jugular vein and brachial artery of healthy volunteers. Calculation of the arteriovenous concentration difference revealed a significant in vivo flux of this steroid from the brain into the circulation in human. Together, these studies identify a novel metabolic route for the elimination of 27-hydroxylated sterols from the brain. Given the emerging connections between cholesterol and neurodegeneration, this pathway may be of importance for the development of these conditions.

A new isotope dilution LC-MS/MS method for assay of 7 alpha-hydroxy-4-cholesten-3-one without need for derivatization is described. This method was used in catheterization experiments on healthy fasting volunteers. The levels of this generally used marker for bile acid synthesis were slightly but significantly higher in the hepatic vein than in the brachial artery. In contrast, the levels of the precursor to 7 alpha-hydroxy-4 cholesten-3-one, 7 alpha-hydroxycholesterol, were the same in the two vessels. It is concluded that there is a net extrahepatic metabolism of 7 alpha-hydroxy-4-cholesten-3-one. The similarity and very high correlation between the levels in the two vessels (r=0.97) are consistent with the contention that 7 alpha-hydroxy-4-cholesten-3-one is a suitable marker for the activity of the hepatic cholesterol 7(x-hydroxylase and thus bile acid synthesis. (C) 2007 Elsevier B.V. All rights reserved.

Recently, we demonstrated a net blood-to-brain passage of the oxysterol 27-hydroxycholesterol corresponding to 4–5 mg/day. As the steady-state levels of this sterol are only 1–2 μg/g brain tissue, we hypothesized that it is metabolized and subsequently eliminated from the brain. To explore this concept, we first measured the capacity of in vitro systems representing the major cell populations found in the brain to metabolize 27-hydroxycholesterol. We show here that 27-hydroxycholesterol is metabolized into the known C27 steroidal acid 7α-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7α-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7α-hydroxy-3-oxo-4-cholestenoic acid in plasma from the internal jugular vein and brachial artery of healthy volunteers. Calculation of the arteriovenous concentration difference revealed a significant in vivo flux of this steroid from the brain into the circulation in human. Together, these studies identify a novel metabolic route for the elimination of 27-hydroxylated sterols from the brain. Given the emerging connections between cholesterol and neurodegeneration, this pathway may be of importance for the development of these conditions.

The blood-brain barrier is almost completely impermeable to cholesterol but allows passage of its side-chain oxidized metabolites. We and others have shown that about 2/3 of the de novo synthesis of cholesterol in this organ is balanced by excretion of 24S-hydroxycholesterol, an oxysterol generated by CYP46A1 in neuronal cells. Another side-chain oxidized cholesterol species, 27-hydroxycholesterol, is taken up by the human brain from the circulation. The latter oxysterol is generated by CYP27A1 in extracerebral tissues, and 27-hydroxycholesterol is thus a link between the extacerebral and intracerebral cholesterol pools. In the brain 27-hydroxycholesterol is rapidly metabolized into 7a-hydroxy-3-oxo-4-cholestenoic acid by the combined action of CYP27A1, CYP7B1 and 3 P-hydroxy steroid dehydrogenase. The C-27-steroid acid is efficiently excreted from the brain. The above conversions are critical for cholesterol homeostasis in the brain with the three cytochrome P-450 species as the most important factors. CYP46A1 and CYP7B1) are present in neuronal cells only and the mechanism for regulation of their activity will be discussed.

Brain cholesterol is eliminated from the brain by conversion into the oxysterol, 24S–hydroxcholesterol, by cholesterol 24–hydroxylase (CYP46A1), which is then fluxed out of the brain into the circulation. In advanced Alzheimer's disease (AD), we have found lower than normal levels of 24S–hydroxycholesterol in the brain, probably due to neuronal loss. In contrast, increased levels of another oxysterol, 27–hydroxycholesterol, were found in AD brain. CYP27A1 which converts cholesterol to 27–hydroxycholesterol is present in the brain but at low levels. To determine if brain 27–hydroxycholesterol originates from the circulation a catheter experiment was carried out on healthy volunteers. Blood samples were collected simultaneously from catheters positioned in the internal jugular vein and brachial artery. 27–Hydroxycholesterol was higher in the arterial samples, demonstrating a net uptake of 27–hydroxycholesterol by the brain. In view of the potent effects of 27–hydroxycholesterol on cholesterol metabolism, this flux is likely to be of importance for intracerebral cholesterol homeostasis. It may also be part of the explanation for the hitherto unexplained link between hypercholesterolemia and Alzheimer's disease. The reason for the net flux of 27–hydroxycholesterol from the circulation into the human brain is unclear, but as we do not observe an accumulation of 27–hydroxycholesterol in healthy brain, this oxysterol must be further metabolized. One proposed mechanism of 27–hydroxycholesterol elimination would involve its metabolism by CYP27A1, CYP7B1 and 3β–hydroxysteroid dehydrogenase, all of which are present in the brain, to form the steroidal acid, 7α–hydroxy–3–oxo–4–cholestenoic acid. Possible metabolites of 27–hydroxycholesterol entering and leaving the brain were measured by GC–MS. Consistent with this hypothesis we found the concentration of 7α–hydroxy–3–oxo–4–cholestenoic acid to be higher in the jugular vein than in the artery, corresponding to a net flux from the brain into the circulation. By incubating 27–hydroxycholesterol with a number of cerebral cell lines, we demonstrated a production of 7α–hydroxy–3–oxo–4–cholestenoic acid in vitro. In view of the potent regulatory effects of 27–hydroxycholesterol on cholesterol homeostasis the present mechanism of elimination may be regarded as a detoxification, and may have a protective role in the development of neurodegenerative diseases.

Side chain oxidized oxysterols have a unique ability to traverse lipophilic membranes. We tested the hypothesis that there is a net flux of 27-hydroxycholesterol from the circulation into the brain using plasma samples collected from the internal jugular vein and an artery of healthy male volunteers. Two independent studies were performed, one in which total levels of 27-hydroxycholesterol were measured and one in which the free fraction of 27-hydroxycholesterol was measured. In the majority of subjects studied, the level of 27-hydroxycholesterol was higher in the artery than in the vein, and uptake from the circulation was calculated to be about 5 mg/24 h. The distribution of 27-hydroxycholesterol in human brain was found to be consistent with an extracerebral origin, with a concentration gradient from the white to the gray matter - a situation opposite that of 24S-hydroxycholesterol, which is exclusively formed in brain. In view of the fact that the blood - brain barrier is impermeable to cholesterol and that 27-hydroxycholesterol is a potent regulator of several cholesterol-sensitive genes, the flux of 27-hydroxycholesterol into the brain may be an important link between intra- and extracerebral cholesterol homeostasis.

Side chain oxidized oxysterols have a unique ability to traverse lipophilic membranes. We tested the hypothesis that there is a net flux of 27-hydroxycholesterol from the circulation into the brain using plasma samples collected from the internal jugular vein and an artery of healthy male volunteers. Two independent studies were performed, one in which total levels of 27-hydroxycholesterol were measured and one in which the free fraction of 27-hydroxycholesterol was measured. In the majority of subjects studied, the level of 27-hydroxycholesterol was higher in the artery than in the vein, and uptake from the circulation was calculated to be about 5 mg/24 h. The distribution of 27-hydroxycholesterol in human brain was found to be consistent with an extracerebral origin, with a concentration gradient from the white to the gray matter

Proinsulin C-peptide was for long considered to be without biological activity of its own. New findings demonstrate, however, that it is capable of eliciting both molecular and physiological effects, suggesting that C-peptide is in fact a bioactive peptide. When administered in replacement doses to animal models or to patients with type 1 diabetes, C-peptide ameliorates diabetes-induced functional and structural changes in both the kidneys and the peripheral nerves. It augments blood flow in a number of tissues, notably skeletal muscle, myocardium, skin and nerve. These effects are thought to be mediated via a stimulatory influence on Na+,K(+)-ATPase and on endothelial nitric oxide synthase. Specific binding of C-peptide to cell membranes of intact cells and to detergent-solubilized cellular components has been demonstrated, indicating the existence of cell-surface binding sites for C-peptide. A number of intracellular responses are elicited by C-peptide, including a rise in Ca2+ concentration and activation of MAP-kinase signaling pathways. Many but not all of C-peptide's intracellular effects can be inhibited by pertussis toxin, supporting the notion that C-peptide may interact via a G-protein-coupled receptor. Additional data suggest that C-peptide may interact synergistically also in the insulin signaling pathway. Combined, the available observations show conclusively that C-peptide is biologically active, even though its molecular mechanism of action is not as yet fully understood. The possibility that replacement of C-peptide in patients with type 1 diabetes may serve to retard or prevent the development of long-term complications should be evaluated.