Biomedical subjects

L Ramos

Publications and source records attributed to L Ramos.

At least 19 recordsLinked to original sources

PubMed2006

Miniaturised selective pressurised liquid extraction of polychlorinated biphenyls from foodstuffs.

The feasibility of miniaturised pressurised liquid extraction (PLE) with in-cell purification and subsequent gas chromatography with micro-electron capture detection (GC-micro-ECD) for the determination of prioritary and toxic polychlorinated biphenyls (PCBs) in a variety of foodstuffs (fat contents in the range 22-49%, w/w, on a freeze-dried basis) has been investigated. After optimisation of the several experimental parameters affecting the efficiency of the selective PLE process, the developed method provided quantitative recoveries of the endogenous PCBs studied and complete fat elimination in a single step using n-hexane as extraction solvent. A total solvent volume of 3.5 mL was used for the two consecutive 7 min static PLEs of 100-mg samples. Detection limits using GC-micro-ECD were below 0.2 ng/g freeze dried sample for all 22 PCBs investigated in real-life foodstuffs, and the repeatability of the complete PLE plus GC-micro-ECD method as calculated for the analysis of the endogenous PCBs in general was better than 14%. Comparison of the miniaturised PLE method developed with either conventional Soxhlet extraction or matrix solid phase dispersion with subsequent (off-line) clean-up for the analysis of non-spiked samples showed that the efficiency of PLE was similar to or better (recoveries in the range 83-133%, as calculated for the endogenous analytes) than for the other two extraction methods assayed.

Analytic Sample Preparation Methods↗

PubMed2006

Globozoospermia revisited.

Globozoospermia is a rare (incidence <0.1%) but severe disorder in male infertility. Total globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa lacking an acrosome. It is still unclear whether patients whose ejaculate contains both normal and globozoospermic cells (partial globozoospermia) suffer from a variation of the same syndrome. Apart from the fact that affected males suffer from reduced fertility or even infertility, no other physical characteristics can be associated with the syndrome. ICSI is a treatment option for these patients, although low fertilization rates after ICSI show a reduced ability to activate the oocyte. In globozoospermic cells, the use of acrosome markers has demonstrated an absent or severely malformed acrosome. Chromatin compaction appears to be disturbed but is not consistently over- or undercondensed. In some cases, an increased number of cells with DNA fragmentation have been observed. The analysis of the cytogenetic composition revealed an increased aneuploidy rate in some cases. Nonetheless, no increased number of spontaneous abortions or congenital defects has been reported in pregnancies conceived after ICSI. The pathogenesis of globozoospermia most probably originates in spermiogenesis, more specifically in acrosome formation and sperm head elongation. In several knockout mouse models, a phenotype similar to that in humans was found. Together with the occurrence of affected siblings, these findings indicate a genetic origin, which makes globozoospermia a good candidate for genetic analysis. More research is needed to elucidate the pathogenesis of human globozoospermia to further understand globozoospermia as well as (abnormalities in) spermiogenesis and spermatogenesis in general.

Adult↗

PubMed2006

Transmission of modified nucleosomes from the mouse male germline to the zygote and subsequent remodeling of paternal chromatin.

Rapidly after gamete fusion, the sperm nucleus loses its specific chromatin conformation and the DNA is repopulated with maternally derived nucleosomes. We evaluated the nature of paternally derived nucleosomes and the dynamics of sperm chromatin remodeling in the zygote directly after gamete fusion. We observed histone H4 acetylated at K8 or K12 already prior to full decondensation of the sperm nucleus, suggesting that these marks are transmitted by the spermatozoon. Tracking down the origin of H4K8ac and H4K12ac during spermiogenesis revealed the retention of nucleosomes with these modifications in the chromocenter of elongating spermatids. We show that sperm constitutive heterochromatin is enriched for nucleosomes carrying specific histone modifications which are transmitted to the zygote. Our results suggest an epigenetic mechanism for inheritance of chromosomal architecture. Furthermore, up to pronucleus formation, histone acetylation and phosphorylation build up in a cascade-like fashion in the paternal chromatin. After formation of the pronucleus, a subset of these marks is removed from the heterochromatin, which suggests a reestablishment of the euchromatin-heterochromatin partition.

Acetylation↗

PubMed2006

Sperm integrity pre- and post-chemotherapy in men with testicular germ cell cancer.

BACKGROUND: While (partial) recovery of spermatogenesis, observed by means of standard semen analysis, has been seen in testicular cancer patients after chemotherapy with cisplatin, sperm genomic integrity and its implication for the patient's fertility are poorly understood. METHODS: Semen and serum from 22 patients treated for testicular cancer were analysed pre- and post-chemotherapy. Besides routine semen analysis, sperm samples were evaluated by computerized karyometric image analysis (CKIA), chromomycin-A3 assay (CMA3, chromatin condensation) and TdT-mediated dUTP nick-end labelling assay (TUNEL, DNA damage). Serum FSH, LH and testosterone concentrations were measured. RESULTS: Ejaculate volume decreased post-chemotherapy (P<0.05). External sperm characteristics (CKIA morphometry) and sperm counts did not deteriorate after chemotherapy. An improvement in DNA condensation was assessed after chemotherapy (37 versus 50% and 47.5 versus 63.7% for CMA3 and CKIA respectively; both P<0.005); yet a high percentage of TUNEL-positive sperm was found in the samples (21 versus 25% for pre- and post-chemotherapy samples respectively). These values were significantly higher than those of a convenience sample of normozoospermic males attending pre-IVF screening. Serum FSH and LH (IU/l) increased after chemotherapy compared with pretreatment levels (8.1 versus 16.7 and 4.5 vs 6.8; both P<0.05, respectively). CONCLUSIONS: Despite the improvement in sperm chromatin packaging after chemotherapy, an abnormally high percentage of DNA-damaged sperm was found in these samples. As sperm quality does not reach normal levels after treatment, it remains difficult to outline the best strategy and guidance concerning fertility potential of testicular cancer patients.

Adult↗

PubMed2006

First 100 liver transplants at the medico-surgical research center (CIMEQ).

In July 1999 we began a liver transplant program that to October 2005 has included 100 liver transplants in 92 patients, eight retransplants, and two combined liver and kidney grafts. Twelve transplants were in pediatric patients, who showed a 92% survival rate in the first year. Fifty-four percent of the patients were male and 46% female. The most frequent etiologies of cirrhosis were 27% hepatitis virus C, 18% alcoholic, 13% cryptogenic, and 11% autoimmune. In 95% of the patients, we preserved the retrohepatic vena cava. The bile duct was reconstructed with a Kehr tube in 46% of patients with 31% using end-to-end suture with a tutor and 15% an end-to-end suture without a tutor. A hepaticojejunostomy was performed in 8%. The immunosuppression included cyclosporine (Neoral) or Prograf combined with mycophenolate mofetil or mycophenolate sodium and steroids. The most frequent biliary complications were local anastomosis leaks (6%) and stenosis (10%). The overall 1-year survival rate was 72%.

Cuba↗

PubMed2006

Solid mesenchymal hamartoma in an adult: a case report.

Mesenchymal hamartoma of the liver (MHL) is an uncommon tumour composed of architecturally abnormal bile ducts in an uncommitted myxoid stroma. Most MHL are diagnosed in childhood and few cases have been reported in adults. This report describes a case of a well defined solid mass in the right lobe of the liver in a 51 year old man. Preoperative radiological examination revealed a large completely solid mass. Biopsy showed a dense fibrous stroma with hyalinisation and some bile ducts. A provisional diagnosis of MHL was made. Surgical excision was impossible and liver transplantation was undertaken. Definitive pathology confirmed the diagnosis. Review of published reports shows this to be the fourth case of MHL treated by liver transplantation.

Hamartoma↗

PubMed2006

The influence of sex steroid hormones on ferrochelatase gene expression in Harderian gland of hamster (Mesocricetus auratus).

Ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the insertion of ferrous iron into protoporphyrin IX to form protohaem. The Syrian hamster Harderian gland (HG) is known for its ability to produce and accumulate large amounts of protoporphyrins. In this species, the female gland contains up to 120 times more porphyrin than the male gland. Data from biochemical studies suggest that this gland possesses the enzymatic complex for haem biosynthesis but lacks ferrochelatase activity. The abundance of intraglandular haem proteins does not support this idea. To gain more insight into this process, we isolated cDNA for ferrochelatase from hamster liver, using the 5'- and 3'- rapid amplification of complementary DNA ends (RACE), and investigated its expression in HG from males and females. The full-length cDNA comprises an open reading frame of 1269 bp encoding a polypeptide of 422 amino-acid residues. Hamster DNA sequence exhibits 92% identity to mouse and 87% identity to human sequences. The predicted hamster enzyme was shown to have structural features of mammalian ferrochelatase, including a putative NH2- terminal presequence, a central core of about 330 amino-acid residues and an extra 30-50-amino-acid stretch at the carboxyl-terminus. RNA blotting experiments indicated that this cDNA hybridized to a liver mRNA of about 2.1 kb, while a weak hybridization signal was observed with mRNA from HG preparations. RT-PCR assays confirmed the expression of specific transcripts in both tissues. Male glands contained approximately twofold more enzyme mRNA than female glands. Likewise, the intraglandular content of mRNA varied during the oestrous cycle, with the highest levels found in the oestrous phase. These cyclic variations were less evident in liver. Ovariectomy plus treatment with progesterone or 17beta-oestradiol plus progesterone increased ferrochelatase mRNA of the gland. In HG of short- or long-term castrated males, the administration of testosterone did not affect the ferrochelatase mRNA concentration. Based on mRNA expression levels, we conclude that Harderian ferrochelatase may play an active role in maintaining the physiological pool of haem required for processing cytochromes and other glandular haem proteins. Likewise, the sex-steroid hormones appear to have only a modest influence upon Harderian ferrochelatase.

Amino Acid Sequence↗

PubMed2005

Liver transplantation at the Cuban Center for Medical and Surgical Research.

From July 4, 1999, when a liver transplantation program was started in Cuba, to October 2003, 66 procedures had been performed in 60 patients. The most frequent reason was cirrhosis caused by hepatitis C virus (29%), and alcoholic cirrhosis (22%). Two patients received simultaneous liver-kidney transplants. Half of the patients were men. Patient ages ranged from 12 to 62 years; the average surgical time was 6 hours; and cold ischemia time was 4 to 14 hours. The average blood consumption was 2033 mL; 2900 mL of plasma and 8 units of platelets were used in 7 cases. Immunosuppression was mainly cyclosporine (Neoral), mycophenolate mofetil or azathioprine, and prednisone. Acute cellular rejections were treated in almost all cases with 3 doses of methylprednisolone. The most frequent complications were biliary (24%), hepatic arterial thrombosis (12%), post-surgical bleeding (10%), acute cellular rejection (24%), and ductopenic rejection (2%). The overall 1-year survival rate was 73.7%.

Cuba↗

PubMed2004

Miniaturization in sample treatment for environmental analysis.

The increasing demand for faster, more cost-effective and environmentally friendly analytical methods is a major incentive to improve the classical procedures used for sample treatment in environmental analysis. In most classical procedures, the use of rapid and powerful instrumental techniques for the final separation and detection of the analytes contrasts with the time-consuming and usually manual methods used for sample preparation, which slows down the total analytical process. The efforts made in this field in the past ten years have led to the adaptation of existing methods and the development of new techniques to save time and chemicals, and improve overall performance. One route has been to develop at-line or on-line and, frequently, automated systems. In these approaches, miniaturization has been a key factor in designing integrated analytical systems to provide higher sample throughput and/or unattended operation. Selected examples of novel developments in the field of miniaturized sample preparation for environmental analysis are used to evaluate the merits of the various techniques on the basis of published data on real-life analyses of trace-level organic pollutants. Perspectives and trends are briefly discussed.

Journal Article↗

PubMed2004

Optimization of a matrix solid-phase dispersion method with subsequent clean-up for the determination of ethylene bisdithiocarbamate residues in almond samples.

A matrix solid-phase dispersion (MSPD) method with subsequent clean-up has been developed to isolate the ethylene bisdithiocarbamate (EBDC) main metabolites (ethylenethiourea, ETU, and ethylenebis [isothiocyanate] sulphide, EBIS) in almond samples. The optimized experimental set-up configuration involved 0.2 g of almond sample, washed sand as MSPD support and NaOH as defatting agent. A subsequent purification step on alumina using acetonitrile as extraction solvent was enough to remove all interferent matrix components, including the fatty material, and provide clean extracts. Quantitative analysis was performed by reversed phase liquid chromatography (RPLC) with diode-array ultraviolet absorbance (DAD UV) detector. Analytes recoveries were between 76 and 85% with relative standard deviations ranging from 3 to 12%. The low limits of quantification of 0.05 and 0.07 mg kg(-1) achieved for ETU and EBIS, respectively, make the method useful for the determination of EBDC residues on almond samples.

Chromatography, Liquid↗

PubMed2004

Levels of polychlorinated biphenyls and organochlorine pesticides in serum samples of Egyptian Vulture (Neophron percnopterus) from Spain.

Concentrations of 23 polychlorinated biphenyls (PCBs), p,p'-DDT and two of its metabolites, p,p'-DDE and p,p'-TDE have been measured in serum samples of up to 1 ml of Egyptian Vulture (Neophron percnopterus) gathered from five populations in Spain. SigmaPCB concentrations were found to be in the range 3.2-97 ng/ml, while those of SigmaDDTs ranged from 0.93 to 38 ng/ml. p,p'-DDT/p,p'-DDE ratios higher than one were only found in the Segovia population, which could be an indication of recent use of p,p'-DDT in the area. In all cases, PCB profiles were dominated by congeners 52, 132 + 105, 138, 153 and 180. However, some differences among the five populations studied became evident when their profiles were compared with those of technical PCB mixtures by principal components analysis. The DDT and PCB levels detected in the serums analysed were lower than those previously reported for similar avian species and those reported to have deleterious effects on survival or reproduction of birds.

Animals↗

PubMed2004

Chromatin condensation and DNA damage of human epididymal spermatozoa in obstructive azoospermia.

Obstructive azoospermia (OA) includes patients with different aetiologies of obstruction such as congenital bilateral absence of vas deferens (CBAVD), post-vasectomy/failed vaso-vasostomy, or unknown (idiopathic). The frequency of DNA-damaged spermatozoa and abnormal chromatin condensation in epididymal spermatozoa of OA patients was investigated for the different aetiology subgroups. DNA damage in caput epididymis spermatozoa was assessed with the TdT-UTP nick-end labelling (TUNEL) assay and chromatin condensation status was measured with chromomycin A3 (CMA3) stain (n = 60 patients). All epididymal sperm samples showed high levels of TUNEL positivity (mean and SD, 43 +/- 17%). CMA3 rates showed large variation within the samples (mean and SD, 53 +/- 21%, range 7-97%); however, a significant difference in chromatin condensation was found between the different aetiologies of obstruction. The highest percentage of CMA3 positivity was found in the CBAVD and idiopathic groups (60 +/- 17 and 65 +/- 18% respectively) compared with the post-vasectomy samples (37 +/- 17%) or ejaculated spermatozoa from normozoospermic males (29 +/- 12%). In conclusion, despite the normal testicular histology in OA men, chromatin condensation differed between OA patients. The physiological heterogeneity found in chromatin condensation between patient subgroups is probably influenced by the aetiology of the obstruction.

Adult↗

PubMed2004

Percutaneous epididymal sperm aspiration: a diagnostic tool for the prediction of complete spermatogenesis.

The classification of azoospermia into obstructive or non-obstructive is largely based on medical history, physical examination and biochemical markers in serum and semen. However, the most accurate parameter for diagnosis is the testicular histology. The predictive value of the percutaneous epididymal sperm aspiration (PESA), FSH, LH, testosterone, inhibin-B and testicular volume was investigated for their accuracy to predict a complete spermatogenesis (Johnsen score > or =8) in order to replace the testicular histology. The specificity and sensitivity of FSH, inhibin-B, LH, testosterone, testicular volume, and the presence of sperm in a PESA procedure was evaluated in 147 azoospermic males attending the centre for infertility diagnosis. A positive PESA outcome presented the highest sensitivity and specificity to predict a Johnsen score > or =8 (93 and 94% respectively) compared with FSH (90 and 19%), inhibin-B (88 and 57%) and testicular volume (95 and 45%). Differences in clinical presentation were observed between patients with positive sperm retrieval with PESA, depending on the aetiology of obstruction. In conclusion, the presence of spermatozoa in the epididymis (PESA+) correlates with a Johnsen score > or =8 and is the most accurate parameter to predict complete spermatogenesis compared with clinical or biochemical parameters. Between obstructive azoospermic patients, the clinical parameters observed varied according to the aetiology.

Biopsy, Needle↗

PubMed2003

[Obstructive azoospermia in men who wish to father children; diagnosis and surgical sperm retrieval].

OBJECTIVE: To evaluate diagnostic procedures and surgical sperm retrieval in men with suspected obstructive azoospermia who wish to father children. DESIGN: Descriptive, retrospective. METHOD: During the period 1 April 1999-31 December 2001 93 men suspected of having obstructive azoospermia underwent surgical sperm retrieval by means of percutaneous epididymal sperm aspiration (PESA). In each patient a testicular biopsy was performed to determine the Johnsen score (a score > or = 8 is equivalent to a normal spermatogenesis). Cryopreservation was performed whenever possible. The findings in both percutaneous and surgical sperm retrieval were compared. RESULTS: In 76 patients (82%) epididymal motile sperm were obtained using PESA. Their Johnsen score on the testis biopsy was 9.1 (range: 7.4-10). In 73 of the patients the Johnsen score was > or = 8. In the 17 patients (18%) in whom no sperm were found with PESA, the median Johnsen score was 5.8 (range: 2-9.8). Epididymal sperm were not found in patients with a testicular volume < 15 ml. In all 28 patients who had undergone a vasectomy in the past, motile sperm were found along with a Johnsen score > or = 8. In 23 of the 24 patients with congenital bilateral absence of the vas deferens (CABVD) the Johnsen score was > or = 8. Cryopreservation was possible in 45 (59%) of all patients and in 5 (35%) of the 13 patients with an unknown cause for the obstructive azoospermia. CONCLUSION: In men with suspected obstructive azoospermia in whom sperm were found using PESA, a diagnostic testis biopsy provided no additional relevant information about the spermatogenesis. There was always a good spermatogenesis after vasectomy. CBAVD patients probably had at least some focal areas in the testes with normal spermatogenesis. Sperm retrieval and cryopreservation could be carried out less frequently in the case of obstructions with an unknown cause.

Adult↗

PubMed2003

[Obstructive azoospermia in men who wish to father children; initial clinical results of intracytoplasmatic sperm injection (ICSI) with surgically retrieved epididymal semen].

OBJECTIVE: To evaluate the results of intracytoplasmatic sperm injection (ICSI) with surgically retrieved epididymal semen. DESIGN: Prospective, descriptive. METHODS: Patients with an obstructive azoospermia confirmed by cytological examination of a testis biopsy, and conforming to the regular IVF/ICSI criteria as laid down in 2001 at the University Medical Centre St Radboud Nijmegen, the Netherlands, were included for percutaneous epididymal sperm aspiration (PESA) and ICSI. The main outcome measure was the ongoing pregnancy rate per initiated cycle. RESULTS: A total of 44 cycles were started in 31 couples. In 43 cases (98%) an ovum pick-up was performed and 41 (93%) embryo transfers were carried out. In 19 (43%) cases, treatment resulted in a positive pregnancy test and 15 (34%) ongoing pregnancies were recorded. In total, 17 healthy children were born (11 boys and 6 girls). CONCLUSION: ICSI with surgically retrieved semen was successfully used as a treatment for childlessness caused by obstructive azoospermia.

Adult↗

PubMed2002

Current use of pressurised liquid extraction and subcritical water extraction in environmental analysis.

This review updates our knowledge about pressurised liquid extraction (PLE) and subcritical water extraction (SWE), two sample preparation techniques which are increasingly used for the extraction of moderately and non-volatile organic pollutants from a variety of solid and semi-solid environmental matrices. Parameters influencing the extraction yield and selectivity are discussed. The results deriving from the analysis of several different classes of compounds in a variety of matrices are compared with a reference method, e.g., Soxhlet extraction. PLE and SWE are both promising techniques due to the short extraction times and low solvent consumption. In addition, SWE offers a wide range of polarities by changing the temperature and can easily provide class-selective extraction by temperature programming and/or the addition of modifier(s). This indicates that, even though many applications have already been reported, more can be expected.

Environmental Monitoring↗

PubMed2002

High-throughput quantification of the anti-leukemia drug STI571 (Gleevec) and its main metabolite (CGP 74588) in human plasma using liquid chromatography-tandem mass spectrometry.

The signal transduction inhibitor STI571 (formerly known as CGP 57148B) or Gleevec received fast track approval by the US Food and Drug Administration (FDA) for treatment of chronic myeloid leukemia (CML). STI571 is a revolutionary and promising new oral therapy for CML, which functions at the molecular level with high specificity. The dramatic improvement in efficacy compared to existing treatments prompted an equally profound increase in the pace of development of Gleevec. The duration from first dose in man to completion of the New Drug Application (NDA) filing was approximately 2.6 years. In order to support all pharmacokinetics studies with sufficient speed to meet various target dates, a semi-automated procedure using protein precipitation was developed and validated. A Tomtec Quadra 96 (Model 320) and a protein precipitation step in a 96-well plate format were utilized. A Sciex API 3000 triple quadrupole mass spectrometer with an atmospheric pressure chemical ionization interface operated in positive ion mode was used for detection. The method proved to be rugged and allowed the simultaneous quantification of STI571 and its main metabolite (CGP 74588) in human plasma. Herein, assay development, validation, and representative concentration-time profiles obtained from clinical studies are presented.

Antineoplastic Agents↗

PubMed2002

Determination of polychlorinated biphenyls in small-size serum samples by solid-phase extraction followed by gas chromatography with micro-electron-capture detection.

An new method for the determination of polychlorinated biphenyls (PCBs) in serum samples of up to 1 ml has been developed. The procedure consisted in the solid-phase extraction (SPE) of the analytes on an Oasis cartridge and the subsequent on-line elimination of the fat by directly dropping of the eluate from the SPE cartridge on a multilayer column placed below the cartridge. This configuration allowed minimising of the sample manipulation as well as the time, solvent and sorbent consumption (i.e. complete sample preparation can be accomplished in about 1 h with only 3 ml of toluene and 300 mg of silica). The SPE plus clean-up method developed showed a satisfactory performance for the analysis of PCBs in rat serum samples providing similar recoveries (i.e. range 73-128% for most of the congeners selected) at the different spiking levels investigated (1.25, 0.50 and 0.25 ng/ml). Detection limits using a microelectron capture detector were in the range 0.01-0.30 ng/ml of serum and the relative standard deviations of the complete method better than 18% irrespective of the PCB concentration. The validated method has been applied to the evaluation for the first time of the PCB levels in serum samples of up to 1 ml from individuals of an Egyptian Vulture colony in Spain.

Animals↗