Plasma hydroxyurea determined by gas chromatography-mass spectrometry.

Hydroxyurea treatment is efficiently used to ameliorate the clinical course of patients affected with sickle cell disease. To understand the patient's wide variation in the clinical response to that drug and monitor its plasma levels, a new method was developed and validated. Fifty microL plasmatic samples containing hydroxyurea are added with internal standard, deproteinized, evaporated to dryness, silanized, and analyzed by gas chromatography-mass spectrometry, which operates in the selected ion mode after electron impact fragmentation. Linearity was found to extend to at least 100mg/L. Over a 1-25mg/L concentration range, coefficients of variation for intra-day and inter-day precision are 5.3% and 7.7%, respectively. Plasma blank-samples reveal endogenous hydroxyurea at a level

In vitro inhibitory effects of disodium cromoglycate on ionic transports involved in sickle cell dehydration.

BACKGROUND: The antiallergic and antiasthmatic drug disodium cromoglycate (DSCG) has also demonstrated an activity against sickle cell disease, but the mechanism of this action still remains unknown. METHODS: Na(+) and K(+) fluxes were studied in red cells obtained from 9 patients affected with sickle cell disease in the absence or in the presence of 1 mM of DSCG and deoxygenated under an N(2) flow during up to 24 h. RESULTS: A significant inhibiting effect of DSCG on the intracellular K(+) exit and the Na(+) entry was observed. CONCLUSION: These results demonstrate that DSCG partially inhibits the abnormal K(+) loss which is implicated in the dehydration of the sickle cell and the stimulation of sickling.

Characterization and comparison of raft-like membranes isolated by two different methods from rat submandibular gland cells.

Lipid rafts are defined as cholesterol and sphingolipid enriched domains in biological membranes. Their role in signalling and other cellular processes is widely accepted but the methodology used for their biochemical isolation and characterization remains controversial. Raft-like membranes from rat submandibular glands were isolated by two different protocols commonly described in the literature; one protocol was based on selective solubilization by Triton X-100 at low temperature and the other protocol consisted in extensive sonication. In both cases a low density vesicular fraction was obtained after ultracentrifugation in a sucrose density gradient. These fractions contained about 20% of total cholesterol but less than 8% of total proteins, and were more rigid than bulk membranes. Fatty acid analyses revealed a similar composition of raft-like membranes isolated by the two different methods, which was characterized by an enrichment in saturated fatty acids in detriment of polyunsaturated acids when compared with the whole cell membranes. Protein profile of detergent resistant membranes or raft-like membranes prepared by sonication was assessed by silver staining after SDS-PAGE and by MALDI-TOF. Both analyses provided evidence of a different protein composition of the Triton X-100 and sonication preparations. Immunoblot experiments revealed that raft-like membranes prepared by detergent extraction or sonication were free of Golgi apparatus or endoplasmic reticulum protein markers (beta-COP and calnexin, respectively) and that they were not substantially contaminated by transferrin receptor (a non-raft protein). While caveolin-1 was highly enriched in raft-like membranes prepared by the two methods, the P2X(7) receptor was enriched in raft-like membrane fractions prepared by sonication, but almost undetectable in the detergent resistant membranes. It can be concluded that both methods can be used to obtain raft-like membranes, but that detergent may affect protein interactions responsible for their association with different membrane domains.

Effect of oral creatine supplementation on urinary methylamine, formaldehyde, and formate.

PURPOSE: It has been claimed that oral creatine supplementation might have potential cytotoxic effects on healthy consumers by increasing the production of methylamine and formaldehyde. Despite this allegation, there has been no scientific evidence obtained in humans to sustain or disprove such a detrimental effect of this widely used ergogenic substance. METHODS: Twenty young healthy men ingested 21 g of creatine monohydrate daily for 14 consecutive days. Venous blood samples and 24-h urine were collected before and after the 14th day of supplementation. Creatine and creatinine were analyzed in plasma and urine, and methylamine, formaldehyde, and formate were determined in 24-h urine samples. RESULTS: Oral creatine supplementation increased plasma creatine content 7.2-fold (P < 0.001) and urine output 141-fold (P < 0.001) with no effect on creatinine levels. Twenty-four-hour urine excretion of methylamine and formaldehyde increased, respectively, 9.2-fold (P = 0.001) and 4.5-fold (P = 0.002) after creatine feeding, with no increase in urinary albumin output (9.78 +/- 1.93 mg x 24 h(-1) before, 6.97 +/- 1.15 mg x 24 h(-1) creatine feeding). CONCLUSION: This investigation shows that short-term, high-dose oral creatine supplementation enhances the excretion of potential cytotoxic compounds, but does not have any detrimental effects on kidney permeability. This provides indirect evidence of the absence of microangiopathy in renal glomeruli.

Effects of disodium cromoglycate on cationic exchange of deoxygenated sickle cells.

In the present work, we explored the way in which cromoglycate, a drug used to treat allergies acts on ion movements in sickle cells. Cells were either slowly deoxygenated by overnight exposure to nitrogen or acutely deoxygenated by exposure to metabisulfite, a strong reducing agent which induces sickling of red blood sickle cells. Flushing the cells with nitrogen increased the intracellular concentration of Na(+) and decreased the intracellular concentration of K(+) and the sum of the concentrations of the two cations. One hundred nM cromoglycate inhibited the decrease of intracellular K(+) and the increase of intracellular Na(+) induced by deoxygenation (n=17). Metabisulfite (100mM) increased the intracellular concentration of Ca(2+) (measured by Fura Red) (n=15) and the shape of the cells (measured by light scattering) (n=9). One µM cromoglycate partially inhibited these two responses. In conclusion, cromoglycate partially inhibits abnormal K(+) loss, Ca(2+) entry pathways or Ca(2+) channels opened by cell deoxygenation and ensuing membrane modifications and prevents cell sickling.

Urinary creatine and methylamine excretion following 4 x 5 g x day(-1) or 20 x 1 g x day(-1) of creatine monohydrate for 5 days.

In this study, we examined the effect of two creatine monohydrate supplementation regimes on 24-h urinary creatine and methylamine excretion. Nine male participants completed two trials, separated by 6 weeks. Participants ingested 4 x 5 g x day(-1) creatine monohydrate for 5 days in one trial and 20 x 1 g x day(-1) for 5 days in the other. We collected 24-h urine samples on 2 baseline days (days 1-2), during 5 days of supplementation (days 3-7), and for 2 days post-supplementation (days 8-9). Urine was assayed for creatine using high-performance liquid chromatography and methylamine using gas chromatography. Less creatine was excreted following the 20 x 1 g x day(-1) regime (49.25 +/- 10.53 g) than the 4 x 5 g x day(-1) regime (62.32 +/- 9.36 g) (mean +/- s; P < 0.05). Mean total excretion of methylamine (n = 6) over days 3-7 was 8.61 +/- 7.58 mg and 24.81 +/- 25.76 mg on the 20 x 1 g x day(-1) and 4 x 5 g x day(-1) regimes, respectively (P < 0.05). The lower excretion of creatine using 20 x 1 g x day(-1) doses suggests a greater retention in the body and most probably in the muscle. Lower and more frequent doses of creatine monohydrate appear to further attenuate formation of methylamine.

Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland.

The plasma membrane of cells from rat submandibular glands was isolated and extensively sonicated. The homogenate was centrifuged at high speed in a discontinuous sucrose gradient. Light fractions contained vesicles analogous to rafts: they were rich in cholesterol, they contained GM1 and caveolin-1, and P2X7 receptors were detected in these fractions. The location of the P2X7 receptors in rafts was abolished when cellular cholesterol was removed by methyl-beta-cyclodextrin (MCD). ATP activated neutral sphingomyelinase (N-SMase), which provoked a decrease of the cellular content of sphingomyelin and an increase of ceramide levels in these cells and in the rafts. Treatment with MCD and filipin (but not with alpha-cyclodextrin) abolished the increase of the intracellular concentration of calcium ([Ca2+]i) in response to epinephrine but not to ATP. MCD and filipin also inhibited the activation by ATP of phospholipase A2 (PLA2). Inhibition of N-SMase with glutathione or GW4869 prevented the activation of PLA2 by P2X7 agonists without affecting [Ca2+]i levels. We conclude that P2X7 receptors are present in both raft and nonraft compartments of plasma membranes; the receptors forming a nonselective cation channel are located in the nonraft fraction. P2X7 receptors in the rafts are coupled to the activation of N-SMase, which increases the content of ceramides in rafts. This may contribute to the activation of PLA2 in response to P2X7 receptor occupancy.

Metabolic, nutritional, iatrogenic, and artifactual sources of urinary organic acids: a comprehensive table.

BACKGROUND: The determination of organic acids and glycine conjugates in urine is key for the diagnosis and follow-up of several inborn errors of metabolism (IEM). However, clinical interpretations may still be hindered by ambiguity in the sources of some urinary organic acids and acylglycines as well as in the relationship between their excretion and IEM. APPROACH: Relevant data have been compiled from major books and references on the topic and by exhaustive bibliographic searches through the Medline and Current Contents databases. CONTENT: A comprehensive table has been designed according to organic acids and conjugates. This table is intended to assist in the interpretation of organic acid profiles because, in addition to IEM, it also refers to other pathologic causes and to physiologic, nutritional, iatrogenic, and artifactual sources. Some preanalytical issues, including possible misinterpretations, are reviewed with regard to IEM.