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PubMed2003

Measurement of total homocysteine in plasma and blood spots using liquid chromatography-tandem mass spectrometry: comparison with the plasma Abbott IMx method.

BACKGROUND: Current sampling for total homocysteine (tHcy) is problematic, requiring plasma separation within 15 min. The aim of this study was to develop a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the measurement of tHcy in plasma and dried blood spots and to determine whether the dried blood spot concentration could be used to predict plasma concentrations of tHcy. METHODS: LC-MS/MS methodology was optimized to measure tHcy in plasma and dried blood spots. Fifty blood samples collected from heart transplant patients were used to form dried blood spots and for plasma analysis. Plasma tHcy was also measured using the Abbott IMx method and values were compared to the tHcy concentrations determined in plasma and dried blood spots using LC-MS/MS methodology. RESULTS: The plasma tHcy LC-MS/MS results compared well with the IMx values: LC-MS/MS=1.18(IMx)-0.44 (r(2)=0.915). The within-batch precision (n =10) of the plasma LC-MS/MS method was < 2.0% at 14.6 and 37.7 micromol/L, respectively; the between-batch precision (n=10) was 5.0 and 8.0%, respectively, at these concentrations. The method was found to be sensitive down to 1 micromol/L and linear up to at least 100 micromol/L. Dried blood spot LC-MS/MS results were considerably lower than the plasma IMx values (P < 0.0001): dried blood spot LC-MS/MS=0.33IMx+1.77 (r(2)=0.682). The within-batch precision (n=20) of the dried blood spot LC-MS/MS method was 7.3% and 4.7% at concentrations of 4.0 and 7.9 micromol/L, respectively; the between-batch precision was 12.6% and 7.9% at concentrations of 5.1 and 8.0 micromol/L, respectively. To assess whether dried blood spots are suitable as a screening test to predict plasma tHcy concentrations, arbitary cut-off levels were compared. If it is assumed that a plasma tHcy concentration of >15 micromol/L is raised, a dried blood spot result of >6.8 micro mol/L has a sensitivity and specificity in detecting a raised plasma tHcy of 83.3% and 96.2%, respectively, and a positive and negative predictive value of 95% and 86%, respectively, with an efficiency of 90%. Use of a dried blood spot cut-off concentration of 6.2 micromol/L for predicting high plasma tHcy concentrations (above 15 micromol/L) has a sensitivity and specificity of 95.8% and 73.1%, respectively, positive and negative predictive values of 76% and 95%, respectively, and an efficiency of 84%. CONCLUSIONS: We have developed a precise and accurate LC-MS/MS method for measuring plasma tHcy concentrations, which uses a small volume of plasma and is suitable for routine use. A satisfactory LC-MS/MS method for the measurement of tHcy in dried blood spots was also developed; this method might be useful in routine screening for raised plasma concentrations of tHcy.

Chemistry, Clinical↗
PubMed1998

Plasma sex hormones are significantly associated with plasma leptin concentration in healthy subjects.

OBJECTIVE: In humans a sexual dimorphism of plasma leptin concentration has been demonstrated but its significance remains to be established. Sex hormones may have a role. PATIENTS: Eighty healthy, non-obese subjects (41M/39F) were studied. MEASUREMENTS: In the whole group of subjects plasma sex hormones, leptin and insulin concentrations were determined, body fat content assessed by bioimpedance analysis, body fat distribution evaluated and insulin-mediated glucose uptake measured by euglycemic hyperinsulinemic glucose. RESULTS: After adjustment for age, gender, amount of body fat, waist/hip ratio (WHR) and fasting plasma insulin concentration, fasting plasma leptin was still significantly correlated with plasma DHEAS (r = -0.30, P < 0.006), oestradiol (r = 0.53, P < 0.001) and testosterone (r = -0.43, P < 0.001) in all subjects (n = 80). Independently of age, amount of body fat and WHR, fasting plasma leptin concentration correlated with plasma oestradiol (r = 0.38, P < 0.01) and total testosterone (r = -0.58, P < 0.001) in males (n = 41) and with fasting plasma oestradiol (r = 0.48, P < 0.002) in females (n = 39). To investigate the independent contribution of anthropometric and hormonal variables to fasting plasma leptin concentration, a multivariate stepwise regression analysis with fasting plasma leptin concentration as dependent variable was made. In the entire group (n = 80), the whole model explained 43% of fasting plasma leptin concentration with fasting plasma insulin, total testosterone and oestradiol concentrations significantly and independently associated with plasma leptin concentration. In this model, fasting plasma DHEAS, testosterone and oestradiol explained 25% of the variability in plasma leptin concentration. CONCLUSION: Our study demonstrates that plasma sex hormone concentrations are associated with plasma leptin concentration.

Analysis of Variance↗
PubMed1989

Fasting plasma caffeine level in cirrhotic patients: relation to plasma levels of catecholamines and renin activity.

Fasting plasma caffeine concentrations, plasma levels of catecholamines and plasma renin activity were measured in patients with cirrhosis and control patients without hepatic dysfunction. A careful dietary history showed no significant difference in caffeine consumption (mean +/- S.E.) among 46 cirrhotics (86 +/- 7 mg per day) vs. 34 control patients (91 +/- 8 mg per day). Fasting plasma caffeine concentrations, however, were significantly higher (7.68 +/- 1.42 micrograms per ml) in cirrhotics than in controls (1.01 +/- 0.20 micrograms per ml) (p less than 0.01). Fasting plasma caffeine concentrations in cirrhotics varied significantly with Child's criteria, namely Child's A patients (2.06 +/- 0.38 micrograms per ml); Child's B patients (6.92 +/- 1.86 micrograms per ml), and Child's C patients (17.70 +/- 3.65 micrograms per ml) (p less than 0.001). In 44 cirrhotics, fasting plasma caffeine concentrations were compared with plasma levels of catecholamines and plasma renin activity. Plasma epinephrine concentrations were normal; however, plasma norepinephrine concentrations were increased in six cirrhotics, and plasma renin activities were increased in 28 cirrhotics. After a 3-day caffeine abstinence, plasma caffeine concentration and renin activity were significantly decreased (p less than 0.01), and high plasma norepinephrine levels were also decreased in 12 cirrhotics. Plasma caffeine concentration, renin activity and norepinephrine level did not change in a control group of cirrhotics who continued to receive caffeine for 3 days (n = 6). After abstinence from caffeine, the decrease of fasting plasma caffeine concentration correlated well with the decrease of plasma renin activity (r = +0.746, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1988

Quantification of the glycolytic origin of plasma glycerol: implications for the use of the rate of appearance of plasma glycerol as an index of lipolysis in vivo.

To assess whether plasma glycerol could be directly derived from plasma glucose, nine postabsorptive dogs were infused with [U-14C] glucose and [2-3H] glycerol to measure the rates of appearance of plasma glucose and glycerol and the conversion of plasma glucose to glycerol before (basal) and after two hours of infusion of glucose (45 mumol/kg/min). Basally (plasma glucose 4.9 +/- 0.2 mmol/L; plasma insulin 5.9 +/- 0.2 microU/mL), rates of appearance of plasma glucose and glycerol were 20 +/- 2 and 5.9 +/- 1.3 mumol/kg/min, respectively, and 1.6 +/- 0.6% of plasma glycerol was derived from plasma glucose. After glucose infusion (plasma glucose 9.1 +/- 0.7 mmol/L; plasma insulin 21.1 +/- 1.9 microU/mL), the rate of appearance of plasma glycerol decreased 80% to 1.1 +/- 0.3 mumol/kg/min and the percent of plasma glycerol from glucose increased significantly to 6.9 +/- 2.9. However, the absolute rate of conversion of glucose to glycerol did not change (0.09 +/- 0.03 v 0.07 +/- 0.03 mumol/kg/min). We conclude that even under conditions of stimulated glycolysis and inhibited lipolysis, only a small amount of plasma glycerol is derived from plasma glucose. Thus, rates of appearance of plasma glycerol can be used as a measure of rates of overall lipolysis in vivo.

Animals↗
PubMed1991

Tissue factor pathway inhibitor with high anticoagulant activity is increased in post-heparin plasma and in plasma from cancer patients.

This study was performed in order to separate plasma fractions of tissue factor pathway inhibitor (TFPI) on the basis of TFPI's heparin binding properties. A main goal was to look for differences in anticoagulant effect of the TFPI fractions from plasma. Normal plasma and plasma with increased amounts of TFPI were used; plasma from cancer patients and post-heparin plasma (plasma drawn 5 min after heparin injection). Heparin affinity chromatography separated plasma TFPI into four fractions with increasing heparin affinity: the flow-through fraction, a low affinity fraction eluting at less than 0.3 M NaCl, an intermediate affinity fraction eluting at 0.3-0.55 M NaCl and a high affinity fraction eluting at 0.55-1.0 M NaCl. These fractions corresponded partly to the three TFPI activity fractions obtained by gel filtration. In plasma from cancer patients, the two fractions with intermediate and high heparin affinity were increased three- to four-fold, compared to normal plasma. The TFPI activity in these two fractions eluted with low-molecular-weight (35-60 kD) on gel filtration. In post-heparin plasma an even larger increase in the fraction with high heparin affinity was found; compared to that in normal plasma it was increased 14-fold. TFPI was purified to 0.72 U/mg protein in this fraction (about 43-fold compared to normal plasma TFPI). The anticoagulant effect of TFPI, relative to the chromogenic substrate TFPI activity, was greater in plasma fractions with high heparin affinity than in the other plasma TFPI fractions, and it was five-fold greater than the anticoagulant effect of recombinant TFPI. Thus, plasma TFPI is heterogenous in heparin affinity and in anticoagulant potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1998

Transport of cholesterol from the endoplasmic reticulum to the plasma membrane is constitutive in CaCo-2 cells and differs from the transport of plasma membrane cholesterol to the endoplasmic reticulum.

The transport of newly synthesized cholesterol from its site of synthesis, the endoplasmic reticulum, to the plasma membrane was studied in CaCo-2 cells. The appearance of newly synthesized cholesterol on the cell surface was rapid. By 30 min, 50% of the total labeled cholesterol was observed in the plasma membrane. The arrival of cholesterol at the plasma membrane was independent of new protein synthesis, a functional Golgi apparatus, or microtubular function. Progesterone, verapamil, and trifluoperazine, inhibitors of p-glycoprotein which are known to inhibit cholesterol transport from the plasma membrane to the endoplasmic reticulum, reduced the amount of newly synthesized cholesterol reaching the plasma membrane. The p-glycoprotein inhibitors, however, caused the accumulation of sterol intermediates in the plasma membrane, suggesting that sterol trafficking to the plasma membrane remained intact, but that trafficking from the plasma membrane to the endoplasmic reticulum was disrupted. In contrast, nigericin, another potent inhibitor of cholesterol movement from the plasma membrane to the endoplasmic reticulum, did not alter the transport of newly synthesized cholesterol to the plasma membrane. Moreover, promoting cholesterol transport from the plasma membrane to the endoplasmic reticulum by sphingomyelin hydrolysis or by micellar cholesterol influx did not alter the percent of newly synthesized cholesterol transported to the plasma membrane. Likewise, preventing plasma membrane cholesterol from reaching the endoplasmic reticulum by incubating cells with lysophosphatidylcholine, filipin, or digitonin did not alter the arrival of newly synthesized cholesterol to the plasma membrane. The results suggest that the amount of cholesterol moving to the plasma membrane from the endoplasmic reticulum is constitutive and regulated at the level of cholesterol synthesis and not at the level of the transport process. The pathways of cholesterol transport to and from the plasma membrane are distinct.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase (III. Is There a Direct Interaction between the Fusicoccin Receptor and the Plasma Membrane H+-ATPase?).

A radioimmunoassay using antibodies raised against bovine serum albumin-conjugated fusicoccin (FC) was applied to measure FC bound to the plasma membrane (PM) isolated from seedlings of radish (Raphanus sativus L.) and of Arabidopsis thaliana treated in vivo plus or minus the toxin. FC bound to the PM from seedlings treated with 5 [mu]M FC was 2-fold (radish) to 7-fold (A. thaliana) higher than the binding capacity of control PM. FC binding depended on the duration of the in vivo treatment but was unaffected by cycloheximide. When FC binding and the PM H+-ATPase activity were compared under different conditions (in vivo or in vitro treatment of different lengths or with different concentrations of FC), a strict linear relation between FC binding and the activation of the PM H+-ATPase was observed in both plant materials under all the conditions tested. Comparison between the maximum binding capacity and the amount of H+-ATPase observed in PM from the two plant materials suggest a one-to-one stoichiometry between the FC receptor and the PM H+-ATPase.

Journal Article↗
PubMed1998

Fusicoccin binding to its plasma membrane receptor and the activation of the plasma membrane H(+)-ATPase. IV. Fusicoccin induces the association between the plasma membrane H(+)-ATPase and the fusicoccin receptor.

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H(+)-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H(+)-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H(+)-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H(+)-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H(+)-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H(+)-ATPase antiserum and with an anti-14-3-3 antiserum indicated an FC-induced association of FCBP with the PM H(+)-ATPase. Analysis of the activation state of PM H(+)-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.

Brassica↗
PubMed1999

Cascade filtration for TTP: an effective alternative to plasma exchange with cryodepleted plasma.

TTP remains enigmatic both in terms of etiology and management. The most recent approach is aggressive plasma exchange (PE) employing cryopoor plasma for replacement, based on the pathogenetic relevance given to exceedingly large Von Willebrand (VWF) multimers in the determination of the syndrome with normalization during remission. PE with fresh frozen plasma (FFP) is better than FFP infusion as shown by a recent Canadian study, supporting the theory that to treat TTP an offending circulating agent needs to be removed from the patient's plasma in contrast to the hypothesis that a missing factor is to be given along with FFP. A more recent hypothesis is supported by the results of studies published by the end of 1998 [Moake J, Chintagumpala M, Turner N et al. Blood 1994;84:490-97; Moake J, McPherson PD. Am J Med 1989;87: 3-9N] which would show that TTP is mediated by auto-antibodies to VWF-cleaving protease, or is the result of deficiency of the protease ascribed to abnormalities in its production, function or survival. Plasmapheresis without plasma infusion is relatively ineffective perhaps because it does not increase the protease activity. Cascade filtration (CF) is the autologous counterpart of plasmapheresis. It has been used by our group since 1980 to remove from patients plasma macromolecules such as VWF, fibrinogen, LDL and circulatory immune complexes (CIC). After secondary filtration, the autologous plasma has a composition which is very similar to that of allogeneic plasma after cryoprecipitation, a product which used in the management of TTP. Based on this knowledge, in 1994 we began to use CF in the treatment of TTP patients. In the beginning (7 patients) CF was combined with a decreasing number of conventional PEs using allogeneic plasma for substitution. Lately only CF with some plasma supplementation has been used in the last 9 cases. From a clinical point of view our 16 patients achieved remission after a number of treatments (11 +/- 7) that compares sufficiently well with those required by our historical control group of 47 cases (14 +/- 13). Of course the patient's exposure to allogenic plasma was significantly lower for patients in the CF only group (1.4 +/- 1.2 plasma U/session) compared to the PE + CF group (4.4 +/- 2.3 plasma U/session) or for the controls treated by PE only (10.8 +/- 4.6 plasma U/session). There were no deaths in the CF or PE + CF groups and no untoward effect was observed. On the contrary there were 5 deaths (1 on the day of presentation) in the PE group, and 1 HBV and 2 HCV infections as well as 4 severe allergic reactions to plasma proteins (or passive antibody infusion). We conclude that CF is presently the best treatment to offer to patients suffering from sporadic TTP and that CF may contribute to expanding the knowledge of the pathogenetic mechanisms of this uncommon multisystem disorder.

Adult↗
PubMed2001

Transfusing methylene blue-photoinactivated plasma instead of FFP is associated with an increased demand for plasma and cryoprecipitate.

BACKGROUND: Photodynamic virus inactivation of plasma with methylene blue significantly decreases the recovery of fibrinogen and coagulation factors V and VIII. Because an adequate supply of fibrinogen is essential for the therapeutic efficacy of transfused plasma in many clinical settings, it was plausible that transfusing photoinactivated plasma (PIP) instead of FFP would result in an increased demand for plasma and cryoprecipitate. STUDY DESIGN AND METHODS: The study involved a retrospective analysis of the use of plasma at a university hospital (Barcelona, Spain) over three 1-year periods: one before the implementation of PIP therapy and two after. Blood components transfused to plasma recipients were listed by broad diagnostic categories based on the Diagnosis-Related Group classification. RESULTS: During the period under study, 2,967 patients were given plasma in this hospital. They received 27,434 units of plasma, 1,660 of cryoprecipitate, 10,079 of platelets, and 24,607 of packed RBCs. Patients undergoing surgical procedures accounted for 74 percent of all transfused plasma. In 71 percent of patient admissions, packed RBCs were transfused in addition to plasma. Diagnostic categories with the greatest requirement for plasma were cardiac valve surgery, liver transplant, wounds and traumatic injuries, and bowel surgery. The use of PIP was associated with a 56-percent increase in the aggregated demand for plasma, whereas the transfusion of non-virus-inactivated cryoprecipitate increased twofold the first year and threefold the second year. The growth in the use of plasma took place in all the diagnostic categories. In those categories that include patients with liver disease, a partial substitution of PIP for cryoprecipitate was observed during the second year after PIP therapy implementation. CONCLUSION: The use of PIP was associated with a marked increase in the demand for plasma and cryoprecipitate, which probably was due to the low hemostatic quality of the new component. It is possible that such an increase overrode the potential health benefits derived from transfusing virus-inactivated plasma.

Aged↗
PubMed1985

Sympathoadrenal activity and plasma glucose effects on plasma dopamine-beta-hydroxylase levels in rats.

Plasma dopamine-beta-hydroxylase (DBH) activity is a controversial index of sympathoadrenal function. In our results, the half-life of bovine DBH administered by cardiac puncture to Wistar rats was dependent on plasma glucose values, being 60 min for controls, 96 min for streptozotocin (STZ)-diabetic animals (p less than 0.02) and 33 min for insulin-treated normal rats (p less than 0.01). In experimental situations with low plasma glucose levels, DBH activity was also diminished with respect to controls (glucose: 103.6 +/- 2.2 mg%, DBH: 9.7 +/- 0.5 U/ml). After fasting, glucose was 60.8 +/- 1.5 mg% (p less than 0.001) and plasma DBH 6.4 +/- 0.3 U/ml (p less than 0.001); fasting plus cold exposure also decreased glucose (66.2 +/- 1.4 mg%, p less than 0.001) and plasma DBH (6.7 +/- 0.2 U/ml; p less than 0.001). In both situations, there was an increase in exocytosis from sympathoadrenal tissues; however, no increase in plasma DBH levels was observed, because plasma glucose being diminished it was unable to compete at the catabolic receptor level. When normal plasma glucose levels take place, plasma DBH is essentially constant, poorly reflecting a moderate increase or decrease in exocytosis from tissues, as was the case in our animals with 48 h of cold exposure. When chemical sympathectomy (6-OH-dopamine) or bilateral adrenalectomy was performed there was a compensatory mechanism between them. Plasma DBH does not change significantly in these situations if plasma glucose values are normal. From these results, the most important physiological influence on plasma DBH activity is the glucose plasma levels. Plasma DBH values not being a useful index of sympathoadrenal activity if, at the same time, the plasma glucose levels are not considered.

Adrenal Glands↗
PubMed1990

Annual cycle of pituitary and plasma gonadotropins and plasma sex steroids in a wild population of the toad, Bufo japonicus.

Pituitary and plasma gonadotropins and plasma sex steroids of free-living toads, Bufo japonicus, were measured monthly from March 1981 to February 1982 and examined in relation to gonadal cycles. Toads were captured at Mizuno, Saitama Prefecture. Individual blood samples were collected by cardiac puncture within 3 min of capture in the field. In males, testicular weight was maximal in August. Plasma follicle-stimulating hormone (FSH) levels changed in association with testicular weight. Plasma androgen levels showed a small peak in November and a large peak in March just prior to breeding. Plasma luteinizing hormone (LH) levels changed in parallel with plasma androgen levels. In females, neither plasma FSH nor LH alone were correlated significantly with ovarian weight. However, ovarian and oviductal weights both correlated significantly with plasma steroid levels. Plasma estradiol levels showed a sharp peak in March, followed by a rapid decrease to the minimum in April. A gradual increase of estradiol occurred from July to November in parallel with an increase in ovarian weight. Changes in plasma progesterone and androgen levels in females resembled those for estradiol. However, the changes in progesterone were not so marked as in estradiol. Plasma androgen levels in females were especially high between January and March. In both sexes, the pituitary gonadotropin contents changed in parallel with plasma levels of both FSH and LH. The pituitary almost always contained more LH than FSH, while the reverse was true in the plasma in both sexes. In addition, plasma FSH levels increased markedly in early summer when plasma LH remained unchanged (males) or increased only slightly (females). These results indicate that the toad may serve as excellent material for the study of differential control of FSH and LH secretion.

Androgens↗
PubMed1988

Immunovisualization of fibrinogen A alpha-chain heterogeneity in normal plasma and plasma from patients with DIC or on streptokinase therapy.

Purified fibrinogen as well as normal plasma, or plasma from patients with DIC or undergoing streptokinase(SK)-therapy was subjected to 1D- and 2D SDS-electrophoresis under reducing conditions. The pattern was revealed either by Coomassie-staining or immunostaining after Western-blotting and then compared. The use of polyclonal antibodies to fibrinogen as well as two monoclonal anti-bodies reacting with FPA and C-terminal part of the A alpha-chain confirmed immunologically the previously reported molecular weight heterogeneity of the A alpha-chain of the fibrinogen molecule as being a constituent of normal plasma, and lead to the following conclusions: 1. The MW-heterogeneity is observed in the fibrinogen pool of normal plasma as well as in DIC-plasma, SK-plasma and in purified fibrinogen, being the least noticeable in normal plasma and most advanced in SK-plasma. Patterns obtained using immunostaining with monoclonal anti-FPA confirm that the MW-heterogeneity of fibrinogen is mainly due to C-terminal degradation of the A alpha-chain. 2. Numerous A alpha-chain remnants (at least 9), with intact N-terminal ends, are found to be present in normal plasma, with a MW range from 66,200 to 36,000 Da, demonstrating that each of the "classical" HMW, LMW, LMW' subgroups consist of fibrinogen molecules which are very heterogeneous. 3. Two populations of A alpha-chains in purified fibrinogen and in fibrinogen in plasma react with the C-terminal specific Mab G-8. This is in contrast to the findings in plasma from streptokinase-treated patients, where several bands of lower molecular weights than the gamma-chain can be seen, suggesting the presence of free, circulating A-alpha chains split in the N-terminal half of the chain beyond the last inter-chain disulphide bond. 4. 2D electrophoresis disclosed substantial deviations in the patterns obtained with DIC-plasma, SK-plasma and with fibrinogen purified by beta-alanine-precipitation from that observed with normal plasma. The present technique allows selective characterization of fibrinogen independently of the other proteins present in plasma and offers extreme sensitivity.

Blotting, Western↗
PubMed1992

Iron-induced ascorbate oxidation in plasma as monitored by ascorbate free radical formation. No spin-trapping evidence for the hydroxyl radical in iron-overloaded plasma.

A study was made of the interaction of plasma ascorbate and ascorbate free radical (AFR) with exogenously added iron. The quantitative determination of AFR has the advantage that transient increases in ascorbate oxidation can be directly monitored by e.p.r. spectroscopy. An AFR signal was found in the plasma of all donors and was unaffected by superoxide dismutase, catalase and the strong iron chelator deferoxamine. These findings and the rapid decrease in AFR under a nitrogen atmosphere suggest that plasma AFR is probably a result of air auto-oxidation. Iron loading of plasma did not affect the intensity of the AFR signal until the iron concentration approached or exceeded the plasma latent iron-binding capacity. In iron-overloaded plasma, the intensity of the AFR signal increased to about 10 times the normal level before decreasing rapidly to undetectable levels after 15-20 min. Determination of plasma ascorbate showed that the disappearance of AFR was due to a complete loss of the vitamin. When 50 microM-ascorbate was loaded with iron in iso-osmotic phosphate buffer there was an increase in the AFR signal, independent of the iron concentration, which was stable at least for 15 min. Thus the rate of ascorbate loss in the iso-osmotic phosphate buffer was considerably lower than in iron-overloaded plasma. The addition of different iron chelators produced comparable effects on the intensity of the AFR signal in both iron-overloaded plasma and ascorbate solution. These results suggest that the characteristic behaviour of plasma AFR after iron loading is due to its specific iron-binding capacity and to plasma ferroxidase activity. The ferroxidase activity of plasma is important to promote the transfer of Fe2+ into transferrin without a transient ascorbate oxidation. Spin-trapping studies with 5,5-dimethyl-1-pyrroline N-oxide and N-t-butyl-alpha-phenylnitrone revealed that iron-overloaded plasma was unable to produce spin-trap adducts even in the presence of 50-300 microM-hydrogen peroxide or 100 microM-azide. Evidence of OH. radical formation was obtained only after the addition of EDTA. Therefore, iron-overloaded plasma itself does not produce a Fenton reaction and, if ascorbate does indeed have a free-radical-mediated pro-oxidant role, it is not detectable in plasma by spin-trapping experiments.

Ascorbic Acid↗
PubMed1998

Acute exogenous elevation of plasma free fatty acids does not influence the plasma magnesium concentration.

OBJECTIVE: Plasma non-esterified (free) fatty acid concentrations rise as a consequence of stimulated endogenous lipolysis and are inversely related to the plasma magnesium concentration when plasma adrenaline concentration is increased. The aim of the study was to test whether high plasma non-esterified fatty acid concentration after infusion of non-esterified fatty acids decreases plasma magnesium concentration. METHODS: Twelve healthy subjects received 500 ml Intralipid or saline in a randomised, cross-over, double-blind design. Infusion of Intralipid results in an isolated elevation of plasma non-esterified fatty acid concentration. Plasma magnesium concentration was determined at baseline and every 30 minutes; plasma non-esterified fatty acid and triglyceride concentrations at baseline and after 120 minutes. RESULTS: Initial plasma magnesium, non-esterified fatty acid, and triglyceride concentrations were similar in both groups. A significant increase in plasma non-esterified fatty acids (2.42 +/- 0.96 mmol/l vs 0.58 +/- 0.23 mmol/l, p = 0.00013) and triglyceride (median and 95th percentile 5.36 (7.35) mmol/l vs 1.18 (1.92) mmol/l, p = 0.003) concentrations was seen with Intralipid. Plasma magnesium concentration increased significantly after Intralipid (0.89 +/- 0.09 mmol/l vs 0.81 +/- 0.06 mmol/l, p = 0.007). No significant changes were seen with saline. A positive association was found between the change in plasma magnesium and triglyceride concentrations (r = 0.85, p = 0.001). CONCLUSION: Acute infusion of non-esterified fatty acids from an exogenous source does not result in a fall in plasma magnesium concentration, indicating that the circulating non-esterified fatty acids play no part in a decrease in plasma magnesium concentration. The high circulating non-esterified fatty acid levels and the fall in plasma magnesium concentration are both a consequence of intracellular lipolysis.

Adult↗
PubMed1987

Immunoradiometric quantitation of tissue plasminogen activator-related antigen in human plasma: crypticity phenomenon and relationship to plasma fibrinolysis.

A two-site immunoradiometric assay for tissue plasminogen activator (tPA) antigen has been developed using immunoaffinity purified antibody. Various treatments enhanced the detection of tPA antigen in the plasma samples. Maximum detection was obtained by acidification of plasma to pH 4.8 to 6.5 or addition of 0.5 mol/L of L-lysine or L-arginine. Acidification or addition of lysine to plasma is also required for maximum immunoadsorption of plasma tPA antigen on anti-tPA-Ig-sepharose. These results indicate that plasma tPA antigen is partially cryptic to antibody in untreated plasma. The plasma tPA antigen isolated by immunoadsorption of either untreated plasma or acidified plasma on anti-tPA-Ig-sepharose consists mainly of a 100-kd plasminogen activator species as determined by fibrin-agar zymography. The 100-kd activity is possibly a tPA:inhibitor complex. A standardized sample preparation method was conveniently adopted by mixing 3 vol of plasma and 1 vol of 2 mol/L of L-lysine for the assay. Reconstitution and recovery studies showed that the method is specific and permits full detection of both free tPA and tPA:inhibitor complex. The validity of the assay is further supported by the finding that the spontaneous plasma fibrinolysis previously demonstrated to be dependent on plasma tPA antigen is correlated with tPA antigen content. Using the standardized assay, we found that tPA antigen concentrations in 16 blood bank plasmas are equivalent to 3.7 to 20 ng of 60 kd tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above.

Acids↗
PubMed2000

Distribution of lipid-soluble antioxidants in lipoproteins from healthy subjects. I. Correlation with plasma antioxidant levels and composition of lipoproteins.

The concentration of five lipid-soluble antioxidants (gamma- and alpha-tocopherol, lycopene, beta-carotene and ubiquinol-10) was measured in plasma and very low-density, low-density and high-density lipoproteins (VLDL, LDL and HDL) isolated from young healthy normo- cholesterolemic subjects. Alpha-tocopherol was the exclusive antioxidant whose plasma concentration significantly correlated with the absolute concentration of total cholesterol (r =0.541, P<0.001). No correlation was found between plasma concentration and lipoprotein content of alpha-tocopherol and ubiquinol-10, whereas it reached statistically significant values for gamma-tocopherol, lycopene and beta-carotene. The alpha-tocopherol content in VLDL and HDL, but not in LDL, was strictly associated with the relative abundance of cholesterol and phospholipids in the lipoprotein particles. Moreover, the difference between alpha-tocopherol concentration in VLDL and LDL appeared to be strictly related to the differences in cholesterol, phospholipids and triglycerides. The percent distribution of the total plasma pool of antioxidant in each lipoprotein class revealed that gamma- and alpha-tocopherol were roughly equally distributed in LDL and HDL. On the other hand, lycopene, beta-carotene and ubiquinol-10 were preferentially sequestered in LDL. Finally, the absolute and relative concentration of alpha-tocopherol, but not that of other antioxidants, in HDL exhibited a statistically significant correlation with plasma HDL/LDL cholesterol ratio. These findings indicate that: (i) plasma concentrations of major lipid-soluble antioxidants are not always predictive of their levels in lipoproteins and that, within individual lipoprotein classes, (ii) the lipid composition, metabolism and relative plasma concentration may significantly affect their abundance.

Adult↗
PubMed1995

Components of biological variation in plasma haptoglobin: relationships to plasma fibrinogen and immune variables, including interleukin-6 and its receptor.

We investigated the components of biological variation, including seasonality, in plasma haptoglobin (Hp) levels and the relationships between plasma Hp and interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6), sIL-2R, fibrinogen (Fb) and absolute number of peripheral blood mononuclear cells, such as leukocytes, neutrophils, monocytes, lymphocytes, CD4+, CD8+, CD25+ T cells and CD20+ B cells. Monthly blood samples were taken from 26 normal volunteers during one calendar year. The estimated inter- and intra-individual C.V. values for plasma Hp were 27.9% and 20.0%, respectively; the index of individuality was 0.72. No significant seasonal rhythms could be detected in plasma Hp levels. The yearly mean values in plasma Hp were significantly and positively related to those in plasma Fb, absolute number of leukocytes, neutrophils, CD4+ T cells and the CD4+/CD8+ T cell ratio. 49.0% of the variance in the yearly mean values of plasma Hp could be explained by variances in serum IL-6 and number of CD4+ (positively related) and CD8+ (negatively related) T cells. There were significant and positive time relationships between plasma Hp, on the one hand, and plasma Fb, sIL-6R, sIL-2R and number of leukocytes, neutrophils and monocytes, on the other. A smaller part of the within-subject variability in plasma Hp (i.e. 6.0%) could be explained by serum sIL-6R and sIL-2R. It is concluded that there are (1) important between-subject differences in the homeostatic setpoints of plasma Hp, which are related to those in plasma Fb and in immune status and (2) significant within-subject, time relationships between plasma Hp and indicators of immune activation and plasma Fb.

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