Biopsy samples were obtained from the vastus lateralis muscle of eight subjects after 0, 20, 60, and 120 s of recovery from intense electrically evoked isometric contraction. Later (10 days), the same procedures were performed using the other leg, but subjects ingested 20 g creatine (Cr)/day for the preceding 5 days. Muscle ATP, phosphocreatine (PCr), free Cr, and lactate concentrations were measured, and total Cr was calculated as the sum of PCr and free Cr concentrations. In five of the eight subjects, Cr ingestion substantially increased muscle total Cr concentration (mean 29 +/- 3 mmol/kg dry matter, 25 +/- 3%; range 19-35 mmol/kg dry matter, 15-32%) and PCr resynthesis during recovery (mean 19 +/- 4 mmol/kg dry matter, 35 +/- 6%; range 11-28 mmol/kg dry matter, 23-53%). In the remaining three subjects, Cr ingestion had little effect on muscle total Cr concentration, producing increases of 8-9 mmol/kg dry matter (5-7%), and did not increase PCr resynthesis. The data suggest that a dietary-induced increase in muscle total Cr concentration can increase PCr resynthesis during the 2nd min of recovery from intense contraction.
The major cholesterol oxidation products in the human circulation are 27-hydroxycholesterol, 24-hydroxycholesterol, and 7␣-hydroxycholesterol. These oxysterols are formed from cholesterol by specific cytochrome P450 enzymes, CYP27, CYP46, and CYP7A, respectively. An additional oxysterol present in concentrations comparable with 7␣-and 24-hydroxycholesterol is 4-hydroxycholesterol. We now report that patients treated with the antiepileptic drugs phenobarbital, carbamazepine, or phenytoin have highly elevated levels of plasma 4-hydroxycholesterol. When patients with uncomplicated cholesterol gallstone disease were treated with ursodeoxycholic acid, plasma 4-hydroxycholesterol increased by 45%. Ursodeoxycholic acid, as well as the antiepileptic drugs, are known to induce cytochrome P450 3A. Recombinant CYP3A4 was shown to convert cholesterol to 4-hydroxycholesterol, whereas no conversion was observed with CYP1A2, CYP2C9, or CYP2B6. The concentration of 4␣-hydroxycholesterol in plasma was lower than the concentration of 4-hydroxycholesterol and not affected by treatment with the antiepileptic drugs or ursodeoxycholic acid. Together, these data suggest that 4-hydroxycholesterol in human circulation is formed by a cytochrome P450 enzyme.Cholesterol oxidation products (oxysterols) have recently attracted great interest because of their numerous biological actions. They have been implicated in bile acid biosynthesis, cholesterol transport, and gene regulation (1). In addition, many oxysterols are toxic to cells and induce apoptosis (2-4). These compounds can be formed either by cholesterol autooxidation or by the action of cholesterol-metabolizing enzymes. Several oxysterols can be formed by both mechanisms, i.e. 7␣-hydroxycholesterol. This oxysterol is a predominant cholesterol auto-oxidation product but is also formed by the hepatic enzyme cholesterol 7␣-hydroxylase. Major oxysterols in the human circulation include 27-hydroxycholesterol, 24-hydroxycholesterol, and 7␣-hydroxycholesterol (5). One additional oxysterol present in human plasma at a relatively high concentration is 4-hydroxycholesterol (6). Very little is known about its formation or metabolism. We have shown earlier that small amounts of this oxysterol are formed, together with 4␣-hydroxycholesterol, during in vitro oxidation of low density lipoprotein, and low levels of the two oxysterols were also found in human atherosclerotic plaques (7). The ratio between 4␣-and 4-hydroxycholesterol was close to one both in oxidized LDL 1 and in plaques, and the amount formed in oxidized LDL was only a small percent of the dominating oxysterol, 7-oxocholesterol. These data suggested that very little 4-hydroxycholesterol is formed by cholesterol auto-oxidation. Because relatively high levels were reported in human plasma we hypothesized that this compound is formed in vivo by an enzymatic reaction. 4␣-and 4-hydroxycholesterol were determined in plasma from volunteers and patients, and it was found that patients treated with certain antiepileptic drugs, known...
Accumulation of amyloid fibrils in the viscera and connective tissues causes systemic amyloidosis, which is responsible for about one per thousand deaths in developed countries1. Localised amyloid can also be very serious, for example cerebral amyloid angiopathy is an important cause of haemorrhagic stroke. The clinical presentations of amyloidosis are extremely diverse and the diagnosis is rarely made before significant organ damage is present1. There is therefore a major unmet medical need for therapy which safely promotes the clearance of established amyloid deposits. Over 20 different amyloid fibril proteins are responsible for different forms of clinically significant amyloidosis and treatments that substantially reduce the abundance of the respective amyloid fibril precursor protein can arrest amyloid accumulation1. Unfortunately control of fibril protein production is not possible in some forms of amyloidosis and in others is often slow and hazardous1. There is no therapy that directly targets amyloid deposits for enhanced clearance. However, all amyloid deposits contain the normal, non-fibrillar, plasma glycoprotein, serum amyloid P component (SAP)2, 3. Here we show that administration of anti-human SAP antibodies to mice with amyloid deposits containing human SAP, triggers a potent, complement dependent, macrophage-derived giant cell reaction which swiftly removes massive visceral amyloid deposits without adverse effects. Anti-SAP antibody treatment is clinically feasible because circulating human SAP can be depleted in patients by the bis-D-proline compound, CPHPC4, thereby enabling injected anti-SAP antibodies to reach residual SAP in the amyloid deposits. The unprecedented capacity of this novel combined therapy to eliminate amyloid deposits should be applicable to all forms of systemic and local amyloidosis.
One of the major oxysterols in the human circulation is 4-hydroxycholesterol formed from cholesterol by the drug-metabolizing enzyme cytochrome P450 3A4. Deuterium-labeled 4-hydroxycholesterol was injected into two healthy volunteers, and the apparent half-life was found to be 64 and 60 h, respectively. We have determined earlier the half-lives for 7␣-, 27-, and 24-hydroxycholesterol to be ϳ0.5, 0.75, and 14 h, respectively. Patients treated with certain antiepileptic drugs have up to 20-fold increased plasma concentrations of 4-hydroxycholesterol. The apparent half-life of deuteriumlabeled 4-hydroxycholesterol in such a patient was found to be 52 h, suggesting that the high plasma concentration was because of increased synthesis rather than impaired clearance. 4-Hydroxycholesterol was converted into acidic products at a much slower rate than 7␣-hydroxycholesterol in primary human hepatocytes, and 4-hydroxycholesterol was 7␣-hydroxylated at a slower rate than cholesterol by recombinant human CYP7A1. CYP7B1 and CYP39A1 had no activity toward 4-hydroxycholesterol. These results suggest that the high plasma concentration of 4-hydroxycholesterol is because of its exceptionally slow elimination, probably in part because of the low rate of 7␣-hydroxylation of the steroid. The findings are discussed in relation to a potential role of 4-hydroxycholesterol as a ligand for the nuclear receptor LXR.4-Hydroxycholesterol is one of the quantitatively most important oxysterols in human circulation (1). We have recently shown that it is formed by the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) 1 (1). Preliminary experiments showed that the formation of this oxysterol by human liver microsomes was relatively slow. The high plasma levels of the oxysterol are therefore surprising, and we hypothesized that this may be a consequence of slow metabolism. Therefore, in this work, we determined the rate of elimination of deuteriumlabeled 4-hydroxycholesterol from plasma. Oxysterols are generally degraded to bile acids, and the rate-limiting step in this conversion is the introduction of a hydroxyl group in the 7␣-position of the steroid. Alternative pathways for bile acid biosynthesis start with oxidation of the steroid side chain by CYP27A1 and CYP46. Therefore, we have studied the possibility that these cytochromes are active toward 4-hydroxycholesterol. The metabolism of 4-hydroxycholesterol was studied in human primary hepatocytes, control, and transfected cells and by incubations with recombinant enzymes. In addition, fecal samples from three untreated subjects and one subject treated with carbamazepine were analyzed for 4-hydroxylated bile acids. Based on these experiments, we present evidence that 4-hydroxycholesterol has an unusually long halflife in plasma and that this is the result of slow elimination, particularly slow 7␣-hydroxylation that is the rate-limiting step for further conversion into bile acids.
Clear differences in the activity of both CYP3A4 and CYP3A5 were shown in the three major human races. Both 4beta-hydroxycholesterol and quinine/3-hydroxyquinine metabolic ratio showed a higher CYP3A activity in women than in men. The results give strong evidence that the plasma concentration of 4beta-hydroxycholesterol may be used as an endogenous marker of CYP3A activity (CYP3A4+5).
In humans, the brain accounts for about 20% of the body's free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated. Here, we describe a novel liquid chromatography (LC) multistage fragmentation mass spectrometry (MS n ) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatization to enhance ionization, exact mass analysis at high resolution to identify potential metabolites, and LC-MS n (n53) to allow their characterization. 24S-hydroxycholesterol was confirmed as a major oxysterol in rat brain, and other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24,-7a,25-, and 7a,27-dihydroxycholesterols. In addition, 3b-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat
It was hypothesized that the reduction of high-energy phosphates in muscle after repeated sprints is smaller in women than in men. Fifteen healthy and physically active women and men with an average age of 25 yr (range of 19-42 yr) performed three 30-s cycle sprints (Wingate test) with 20 min of rest between sprints. Repeated blood and muscle samples were obtained. Freeze-dried pooled muscle fibers of types I and II were analyzed for high-energy phosphates and their breakdown products and for glycogen. Accumulation of plasma ATP breakdown products, plasma catecholamines, and blood lactate, as well as glycogen reduction in type I fibers, was all lower in women than in men during sprint exercise. Repeated sprints induced smaller reduction of ATP and smaller accumulation of IMP and inosine in women than in men in type II muscle fibers, with no gender differences in changes of ATP and its breakdown products during the bouts of exercise themselves. This indicates that the smaller ATP reduction in women than in men during repeated sprints was created during recovery periods between the sprint exercises and that women possess a faster recovery of ATP via reamination of IMP during these recovery periods.
Farnesoid X receptor knockout (Fxr 2/2 ) mice cannot upregulate the bile salt export pump in bile acid loading or cholestatic conditions. To investigate whether Fxr 2/2 mice differ in bile acid detoxification compared with wild-type mice, we performed a comprehensive analysis of bile acids extracted from liver, bile, serum, and urine of naive and common bile duct-ligated wild-type and Fxr 2/2 mice using electrospray and gas chromatography mass spectrometry. In addition, hepatic and renal gene expression levels of Cyp2b10 and Cyp3a11, and protein expression levels of putative renal bile acid-transporting proteins, were investigated. We found significantly enhanced hepatic bile acid hydroxylation in Fxr 2/2 mice, in particular hydroxylations of cholic acid in the 1b, 2b, 4b, 6a, 6b, 22, or 23 position and a significantly enhanced excretion of these metabolites in urine. The gene expression level of Cyp3a11 was increased in the liver of Fxr 2/2 mice, whereas the protein expression levels of multidrug resistance-related protein 4 (Mrp4) were increased in kidneys of both genotypes during common bile duct ligation. In conclusion, Fxr 2/2 mice detoxify accumulating bile acids in the liver by enhanced hydroxylation reactions probably catalyzed by Cyp3a11. The metabolites formed were excreted into urine, most likely with the participation of Mrp4.-Marschall, H-U.,
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