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Biomedical subjects

M A Garcia

Publications and source records attributed to M A Garcia.

At least 19 recordsLinked to original sources
PubMed1999

Determination of marbofloxacin in plasma samples by high-performance liquid chromatography using fluorescence detection.

A simple and sensitive HPLC method has been developed for the determination of marbofloxacin (MAR) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. MAR and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column and eluted with aqueous solution-acetonitrile (80:20). The fluorescence of the column effluent was monitored at lambda(ex) = 338 and lambda(em) = 425 nm. The retention times were 2.20 and 3.30 min for MAR and ENR, respectively. The method was shown to be linear from 15 to 1500 ng/ml (r2 = 0.999). The detection limit was 15 ng/ml. Mean recovery was determined as 90% by the analysis of plasma standards containing 150, 750, and 1500 ng/ml. Inter- and intra-assay precisions were 3.3% and 2.7%, respectively.

Anti-Infective Agents↗
PubMed1999

Regulation of intracellular pH in capacitated human spermatozoa by a Na+/H+ exchanger.

We previously demonstrated that the progesterone-(P) initiated human sperm acrosome reaction (AR) was dependent on the presence of extracellular Na+ (Na(-)0). Moreover, Na(-)0 depletion resulted in a decreased cytosolic pH (pHi), suggesting involvement of a Na(+)-dependent pHi regulatory mechanism during the P-initiated AR. We now report that the decreased pHi resulting from Na(+)0 depletion is reversible and mediated by a Na+/H+ exchange (NHE) mechanism. To determine the role of an NHE in the regulation of pHi, capacitated spermatozoa were incubated in Na(+)-deficient, bicarbonate/CO2-buffered (ONaB) medium for 15-30 min, which resulted in an intracellular acidification as previously reported. These spermatozoa were then transferred to Na(+)-containing, bicarbonate/CO2-buffered (NaB) medium; Na(+)-containing, Hepes-buffered (NaH) medium; or maintained in the ONaB medium. Included in the NaH medium was the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA). The steady-state pHi was then determined by spectrofluorometric measurement of bis(carboxyethyl)5(6)-carboxyfluoroscein (BCECF) fluorescence. EIPA (0.1 microM) significantly (P < 0.05) inhibited the pHi recovery produced by NaH medium. Moreover, the pHi in NaH medium was not significantly (P < 0.05) different than NaB medium. These results indicate that a Na(+)-dependent, bicarbonate-independent pHi regulatory mechanism, with a pharmacological characteristic consistent with an NHE, is present in capacitated spermatozoa. In support of the involvement of a sperm NHE, we also demonstrated specific immunoreactivity for a 100 kDa porcine sperm protein using an NHE-1 specific monoclonal antibody. Interestingly, no significant (P = 0.79) effect was seen on the P-initiated AR when EIPA was included in either the NaH or NaB medium. While these findings suggest that inhibition of NHE-dependent pHi regulation in capacitated spermatozoa is not sufficient to block initiation of the AR by P, they do not preclude the possibility that an NHE mediates the regulation of capacitation or sperm motility.

Acrosome↗
PubMed1999

Simultaneous determination of enrofloxacin and its primary metabolite, ciprofloxacin, in plasma by HPLC with fluorescence detection.

A simple and sensitive HPLC method has been developed for the simultaneous determination of enrofloxacin (ENR) and ciprofloxacin (CIP) in plasma. Plasma sample preparation was carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. ENR, CIP and the internal standard, sarafloxacin (SAR), were separated on a reversed-phase column, and eluted with aqueous acetonitrile (80:20). The fluorescence of the column effluent was monitorized at lambda(ex) 338 and lambda(em) 425 nm. The retention times were 2.28, 3.30 and 4.40 min for CIP, ENR and SAR, respectively. The detection limit for the two compounds was 10 ng/mL. Standard curves were linearly related to concentration in the range from 1 to 1500 ng/mL. The recovery was 93% for ENR and 75% for CIP.

Anti-Infective Agents↗
PubMed1999

Diagnosis of secondary myelodysplastic syndromes (MDS) following autologous transplantation should not be based only on morphological criteria used for diagnosis of de novo MDS.

Secondary myelodysplastic syndromes (MDS) are increasingly being reported after autologous transplantation. Transient dysplastic changes have also been observed after this type of treatment. However, to the best of our knowledge no systematic morphological analysis has been performed to determine the influence of stem cell transplantation on bone marrow morphology. In 53 patients undergoing autologous transplantation, we evaluated the bone marrow, before and 6 and 12 months after the transplant, in order to analyze the appearance of dyshemopoietic changes, assessed according to a pre-established score. We also studied 25 bone marrow samples obtained at the time of diagnosis, prior to treatment, but we did not find morphological atypia. Six months after transplant, cellularity and thrombopoiesis had decreased in 38% and 49% of patients respectively, although 1 year after the process they were normal in most cases. Myelodysplasia was already present in bone marrow before transplantation and continued to be in evidence for a long time afterwards. This suggests that chemotherapy and radiotherapy used prior to transplantation are responsible for dysplastic changes. The myeloid line was the most affected with 100% of patients showing dysgranulopoiesis 1 year after autografting. Cytopenias were observed in 51% and 44% of patients 6 and 12 months after transplantation. Moreover, concomitant presence of cytopenia and myelodysplasia was observed in 37.7% of patients at 6 months after transplantation and 25% at 12 months, and therefore they could be diagnosed with MDS. These data contrast with the incidence of secondary MDS reported in earlier publications. According to these findings, the value of the French-American-British Co-operative Group criteria for the diagnosis of MDS following autologous transplantation is questionable. Moreover, since dyshemopoietic features are almost always present after autologous transplant, morphological criteria are not useful for early recognition of patients with secondary MDS after transplantation.

Adult↗
PubMed1999

Progesterone-mediated calcium influx and acrosome reaction of human spermatozoa: pharmacological investigation of T-type calcium channels.

The mechanisms of the progesterone (P4)-activated Ca2+ influx and the relationship between the intracellular free Ca2+ concentration ([Ca2+]i) and the acrosome reaction (AR) were investigated in this study. We compared the [Ca2+]i of uncapacitated and capacitated human sperm populations in response to P4 stimulation; characterized the effects of the pharmacological agents pimozide and mibefradil, inhibitors of T-type voltage-operated calcium channels (VOCCT), on the P4-activated Ca2+ influx; and determined the effects of these drugs on the P4-initiated AR. Since pimozide can also inhibit calmodulin-dependent enzymes, we examined the effects of the calmodulin antagonist, calmidazolium, on the above-mentioned events. The basal [Ca2+]i and the amplitude of the P4-activated Ca2+ influx were significantly (p < 0.05) higher in capacitated sperm populations. Also, in capacitated sperm populations, all three pharmacological agents significantly (p < 0.05) inhibited the P4-activated Ca2+ influx (IC50): calmidazolium (0.7 microM) > pimozide (8 microM) > mibefradil (11 microM). By contrast, the effects of these drugs on the P4-initiated AR were varied: pimozide (10 and 20 microM) significantly (p < 0.05) increased the percentage of AR spermatozoa, calmidazolium was without effect, and mibefradil (20 microM) significantly (p < 0.05) inhibited the AR. These disparate results do not allow us to reach any definitive conclusion concerning the role of a sperm VOCCT in the mechanism of the P4-initiated AR. However, the differences between the [Ca2+]i and AR effects, in particular the inverse relationship in the case of pimozide, suggest a dissociation between the amplitude of the P4-stimulated Ca2+ signal and the downstream biological effect of that signal, the AR.

Acrosome Reaction↗
PubMed1998

Lack of effect of walking on labor and delivery.

BACKGROUND AND METHODS: Walking during labor may reduce patients' discomfort and improve outcomes. We conducted a randomized trial of walking during active labor to determine whether it altered the duration of labor or other maternal or fetal outcomes. Women with uncomplicated pregnancies between 36 and 41 weeks' gestation and in active labor were randomly assigned either to walking or to no walking (usual care). Pedometers were used to quantify walking, and the time spent walking was recorded. RESULTS: Of the 536 women assigned to the walking group, 380 actually walked. Their mean (+/-SD) walking time was 56+/-46 minutes. There were no significant differences between the women assigned to the walking group and the 531 women assigned to the usual-care group in the duration of the first stage of labor (6.1 hours in both groups, P=0.83), the need for labor augmentation with oxytocin (23 percent vs. 26 percent, P=0.25), and the use of analgesia (84 percent vs. 86 percent, P=0.59). Similarly, the percentages of women requiring delivery by forceps (4 percent vs. 3 percent, P=0.35) and cesarean section (4 percent vs. 6 percent, P=0.25) were not significantly different. These labor and delivery outcomes were unrelated to walking in both nulliparous and parous women. The infants' outcomes were also similar in the two study groups. CONCLUSIONS: Walking neither enhanced nor impaired active labor and was not harmful to the mothers or their infants.

Adult↗
PubMed1998

Cutaneous injuries in women athletes.

Positioned at the interface between the athlete and her sports environment, the skin bares the brunt of multiple forces, many of which result in cutaneous trauma. Although this subject has been reviewed in considerable detail, we wish to focus on those injuries which are unique to women competitors and fitness advocates either exclusively, or more commonly, with equal or greater frequency than their male counterparts. We hope that these topics will be of value to dermatology nurses in answering questions on the subject both in and out of the clinical setting.

Athletic Injuries↗
PubMed1997

Simultaneous determination of verapamil and norverapamil in biological samples by high-performance liquid chromatography using ultraviolet detection.

In this paper we develop an high-performance liquid chromatographic method with ultraviolet detection for the determination of verapamil and its primary metabolite norverapamil in biological samples. Both compounds, as well as the internal standard, imipramine, were extracted from alkalinised blood, with n-hexane-isobutyl alcohol, back-extracted into 0.01 M phosphoric acid and determined using a reversed-phase column and ultraviolet monitoring at 210 nm. The average coefficient of variation obtained over the concentration range of 1-1000 ng/ml is about 3%. The detection limit is below 5 ng/ml for both compounds, and extraction recoveries close to 80%. The method was applied to a pharmacokinetic study of the drug and its active metabolite and used to analyse blood samples from verapamil treated rabbits.

Animals↗
PubMed1997

Affinity chromatography of RNase inhibitor.

Of the reagents used in reverse transcription-PCR (RT-PCR), RNase Inhibitor is the most costly on a per assay basis. A simple method for purification of RNase Inhibitor from bovine liver is described. Approximately 40,000 units of RNase Inhibitor can be purified to homogeneity from 400 g of bovine liver within two days using inexpensive reagents. The method described employs (a) facile procedures to rapidly obtain a clarified liver extract, (b) affinity chromatography of RNase Inhibitor on RNase-A-Sepharose, and (c) batchwise concentration of the purified protein with a molecular sieve resin.

Animals↗
PubMed1997

The toxic and accumulative effects of short-term exposure to cadmium in rainbow trout (Oncorhynchus mykiss).

Groups of 20 trout (Oncorynchus mykiss) were exposed for 3-w periods to different subacute oral doses of Cl2Cd (0.01, 0.025 or 0.05 microgram Cd/mL). Cadmium toxicity and accumulation were studied in kidney, liver, gills, muscle and brain weekly. Cadmium determination was carried out using atomic absorption spectrocopy (AAS) with graphite furnace. After 21 d exposure, cadmium concentrations increased significantly. In all tissues studied except muscle and brain. Cadmium storage presented the following sequence: kidney > liver > gills. At the beginning, cadmium accumulation was linearly dose dependent (r = 0.999). Then, at the highest dose (0.05 microgram Cd/mL) accumulation increased rapidly during the second week of exposure until equilibrium was reached. In contrast, liver did not accumulate cadmium selectively, suggesting that liver is able to eliminate cadmium, perhaps through metallothionein formation. Cadmium levels in gills were higher than controls (P < 0.05) and showed signs of equilibrium after the first week. According to organ weight/body weight ratios, there was similar accumulation behavior. Liver and kidney ratios increased dose dependently, but gills and brain did not have significant differences.

Absorption↗
PubMed1996

Importance of sodium ion to the progesterone-initiated acrosome reaction in human sperm.

Progesterone (P) has previously been shown to rapidly increase free intracellular calcium concentration ([Ca2+]i), and subsequently to initiate the acrosome reaction (AR) in capacitated human sperm. The present study used cytochemical analysis of the AR, and spectrofluorometric determination of sperm [Ca2+]i and intracellular pH (pHi) in Na(+)-containing and Na(+)-deficient bicarbonate/CO2-buffered media to investigate the role of Na+ in these P-initiated changes. We found that P failed to initiate the AR in Na(+)-deficient medium, and that the initial rise in [Ca2+]i following P (1 microgram/ml) stimulation was similar for both media; however, the [Ca2+]i in the Na(+)-deficient medium regressed more rapidly and plateaued at a significantly lower [Ca2+]i. Moreover, the differences in plateau [Ca2+]i were directly related to the percentage of acrosome reactions, suggesting that the plateau phase is not due to [Ca2+]i, but rather to the release of intracellular fura-2 into the medium during the AR. These [Ca2+]i, and AR results are in contrast to those reported previously by others for human sperm and suggest that a Na(+)-dependent mechanism is important in the P-initiated human sperm AR. Such a Na+ requirement may reflect the involvement of this ion in pHi regulation, as capacitated sperm that were incubated in a Na(+)-deficient medium for > or = 30 min displayed a significantly lower pHi.

Acrosome↗
PubMed1996

RT-PCR without RNA isolation.

Reverse transcription-PCR (RT-PCR) has traditionally required time-consuming RNA extraction and purification. This report demonstrates that one can completely avoid the RNA extraction step in RT-PCR by basing the comparison of samples on cell number rather than micrograms of total RNA. A new method for lysing cells while preserving RNA is described. RT-PCR is carried out (i) by rapidly freezing cells in the presence of ribonuclease inhibitor (RNase inhibitor) plus dithiothreitol and (ii) by using extracts of 250 or fewer cells directly in the RT-PCR assay. Aldolase mRNA, extracted by freeze-thawing cells in the presence of RNase inhibitor, was found to be stable at 42 degrees C for over three hours. Since the RT step can be completed within 1 h, there is minimal degradation of mRNA. This simple procedure avoids the use of harsh reagents, which may inhibit enzymes involved in RT-PCR, and produces results virtually identical to methods that employ guanidinium thiocyanate and phenol for RNA extraction. Optimized conditions for each parameter of the procedure are described that permit amplification of mRNA from as few as four cells.

Freezing↗
PubMed1995

DNA aneuploidy in acute myeloblastic leukemia is associated with a high expression of lymphoid markers.

In the present study the DNA cell content of 205 de novo acute myeloblastic leukemia (AML) patients is analyzed at flow cytometry in order to determine both the incidence of DNA aneuploidy in AML patients and the clinical and biological characteristics of AML aneuploid cases. All technical procedures were performed in accordance with the proposed guidelines of the DNA Cytometric Consensus Conference. Our results show that the incidence of DNA aneuploidy is quite low (4.8%), with most cases (n = 8) hyperdiploid and only a small proportion (n = 2) hypodiploid. No major differences were detected between the aneuploid and diploid cases with respect to the clinical and prognostic disease characteristics. Regarding the immunophenotype of the blast cells, the aneuploid cases displayed both a higher incidence of immature myeloblastic phenotype and a greater expression of lymphoid-associated antigens. In summary, our results show that AML patients display a quite low incidence of DNA aneuploidy and that despite the fact that these cases do not display particular clinical characteristics, they show an association with the expression of lymphoid-related markers.

Adult↗
PubMed1995

Program planning: a total quality approach.

1. Deming's concepts for total quality management/continuous quality improvement are the basis for a tool occupational health nurses can apply in health program planning. 2. The program planning model provides a framework for systematically implementing, evaluating, and improving products and services. In the current business environment, this is critical to success and to demonstration of the value of occupational health services to a company. 3. A case study describing implementation of a medical case management program illustrates application of the model in a real life situation.

Humans↗
PubMed1994

Comparison of derivatization procedures for the determination of diuretics in urine by gas chromatography-mass spectrometry.

Three different GC-MS screening procedures, which use different ways of derivatization (methylation), are compared. In the first one, derivatization with iodomethane in acetone and a previous solid-liquid extraction is used; the second one is based on an extractive alkylation method using iodomethane in toluene (liquid-liquid extraction); and the last one is flash methylation by pyrolysis of tetraalkylammonium salts in the injector of the gas chromatograph using trimethylanilinium as the derivatization agent. The speed of the extraction, reproducibility and accuracy have been compared for 20 diuretics including the ones most often used in sports, such as bumetanide, ethacrynic acid, acetazolamide, dichlorphenamide, furosemide, hydroflumethiazide, hydrochlorothiazide and chlorthalidone; they have also been applied to the two uricosuric agents probenecid and benzbromarone.

Diuretics↗
PubMed1994

Progesterone initiation of the human sperm acrosome reaction: the obligatory increase in intracellular calcium is independent of the chloride requirement.

The progesterone-initiated human sperm acrosome reaction (AR) requires a rise in intracellular Ca2+ ([Ca2+]i), extracellular Cl- and apparently increased Cl- flux through a unique steroid receptor/Cl- channel resembling but not identical to a GABA(A)/Cl- channel complex. The present study uses fura-2 loaded human sperm, GABA(A)/Cl- channel blockers (picrotoxin and pregnenolone sulfate) and Cl(-)-containing and Cl(-)-deficient media to determine whether the progesterone-mediated increase in [Ca2+]i is dependent on the Cl- requirement. There was no significant difference between the progesterone-mediated increases of [Ca2+]i obtained in Cl(-)-containing and Cl(-)-deficient media. Picrotoxin did not significantly inhibit the progesterone-mediated increase in [Ca2+]i, and pregnenolone sulfate increased [Ca2+]i to the same extent as progesterone. These results strongly suggest that the increase in [Ca2+]i essential to the AR is independent of the AR Cl- requirement and could be explained by the existence of two different sperm plasma membrane progesterone receptors.

Acrosome↗
PubMed1994

Comparative evaluation of pentoxifylline, buflomedil, and nifedipine in the treatment of intermittent claudication of the lower limbs.

In the light of the current controversy surrounding the use of hemorheologic and vasodilator drugs in the treatment of peripheral arteriosclerosis, a comparative study was designed in order to evaluate the efficacy of pentoxifylline, buflomedil, and nifedipine in 45 patients with peripheral arterial disease (Fontaine stage II). The patients in this prospective randomized study were divided into three groups: 15 patients received pentoxifylline treatment (1,200 mg/day), 15 were treated with buflomedil (600 mg/day), and 15 with nifedipine (60 mg/day). Response to treatment was assessed at the start of the study and after forty-five and ninety days, by clinical examination, Doppler test, strain test, and digital occlusion plethysmography using a strain gauge ring. Pentoxifylline was significantly more effective (P < 0.05) than buflomedil and nifedipine at ninety days in improving walking performance, resting toe pressure, resting and postexercise ankle/brachial pressure ratio, and basal/postischemic toe-pulse ratio. Significant differences within groups were also noted for initial claudication, toe peak-flow time, pulse reappearance time (PRT/2), and maximum postischemic flow time, together with significant intergroup variables. In conclusion, pentoxifylline proved more effective than the other drugs tested in: 1. improving distal pressure and resting microcirculatory blood flow; 2. increasing postexercise distal flow, ratios, and pressures and enabling faster recuperation of basal pulse rates; 3. increasing initial claudication distance in the strain test within the test group and achieving a greater absolute subjective claudication distance than that obtained using the other treatments.

Adult↗