HPLC determination of Vitamin D(3) and its metabolite in human plasma with on-line sample cleanup.

A HPLC method with automated column switching and UV-diode array detection is described for the simultaneous determination of Vitamin D(3) and 25-hydroxyvitamin D(3) (25-OH-D(3)) in a sample of human plasma. The system uses a BioTrap precolumn for the on-line sample cleanup. A sample of 1ml of human plasma was treated with 2ml of a mixture of ethanol-acetonitrile (2:1 (v/v)). Following centrifugation, the supernatant was evaporated to dryness under a stream of dry and pure nitrogen. The residue was reconstituted in 250muL of a solution of methanol 5mmoll(-1) phosphate buffer, pH 6.5 (4:1 (v/v)), and a 200mul aliquot of this solution was injected onto the BioTrap precolumn. After washing during 5min with a mobile phase constituted by a solution of 6% acetonitrile in 5mmoll(-1) phosphate buffer, pH 6.5 (extraction mobile phase), the retained analytes were then transferred to the analytical column in the backflush mode. The analytical separation was then performed by reverse-phase chromatography in the gradient elution mode with the solvents A and B (Solvent A: acetonitrile-phosphate buffer 5mmoll(-1), pH 6.5; 20:80 (v/v); solvent B: methanol-acetonitrile-tetrahydrofuran, 65:20:15 (v/v)). The compounds of interest were detected at 265nm. The method was linear in the range 3.0-32.0ngml(-1) with a limit of quantification of 3.0ngml(-1). Quantitative recoveries from spiked plasma samples were between 91.0 and 98.0%. In all cases, the coefficient of variation (CV) of the intra-day and inter-day-assay precision was

[Effect of cooper supplementation on blood pressure values in patients with stable moderate hypertension]. / Efecto de la suplementación con cobre sobre los valores de presión arterial en pacientes con hipertensión moderada estable.

Copper (Cu) deficiency is associated with changes in arterial pressure. The effect depends of the age of initiation of the copper-deficient diet. Copper deficiency started at a young age causes hypotension. When initiated in older or adult animals, copper deficiency can cause hypertension. A case-control study was carried out to investigate the effect of administrating 5 mg Cu/d in 60 subjects, both genders, with mild stable hypertension, pharmacologically untreated (treated group) and compared with 60 hypertensives (control group) who were matched by gender, age, body weight, smoking habits, calories, fat and salt intake (NaCl), and physical activity. Hypertension was diagnosed when the blood pressure was > 150/95 mm Hg. Mean age, mean corporal weight and risk factors were similar in both groups. The results suggested the existence of a marginal deficiency of the trace element in 62% of subjects and demonstrated that Cu decreases systolic (r = -0.963) and diastolic (r = -0.981) blood pressures in treated group (p < 0.05). Control patients did not show significant changes in their arterial pressures. These findings indicate a functional alteration in human blood pressure regulation during mild copper depletion and suggest that Cu could be used in the treatment of stable moderate arterial hypertension. Further investigation is needed to determine the extent of this influence.

Efecto de la suplementación con cobre sobre los valores de presión arterial en pacientes con hipertensión moderada estable / Effect of cooper supplementation on blood pressure values in patients with stable moderate hypertension

Copper (Cu) deficiency is associated with changes in arterial pressure. The effect depends of the age of initiation of the copper-deficient diet. Copper deficiency started at a young age causes hypotension. When initiated in older or adult animals, copper deficiency can cause hypertension. A case-control study was carried out to investigate the effect of administrating 5 mg Cu/d in 60 subjects, both genders, with mild stable hypertension, pharmacologically untreated (treated group) and compared with 60 hypertensives (control group) who were matched by gender, age, body weight, smoking habits, calories, fat and salt intake (NaCl), and physical activity. Hypertension was diagnosed when the blood pressure was > 150/95 mm Hg. Mean age, mean corporal weight and risk factors were similar in both groups. The results suggested the existence of a marginal deficiency of the trace element in 62 per cent of subjects and demonstrated that Cu decreases systolic (r = -0.963) and diastolic (r = -0.981) blood pressures in treated group (p < 0.05). Control patients did not show significant changes in their arterial pressures. These findings indicate a functional alteration in human blood pressure regulation during mild copper depletion and suggest that Cu could be used in the treatment of stable moderate arterial hypertension. Further investigation is needed to determine the extent of this influence.

Serum trace elements and fat-soluble vitamins A and E in healthy pre-school children from a Venezuelan rural community.

Zn (SZn), Cu (SCu), Fe (SFe), vitamin A (SVA) and vitamin E (SVE) were measured in blood serum samples of 85 healthy pre-school children aged 2-6 yr. from the rural community of Canaguá, Mérida State, Venezuela. The relationship between these biochemical indexes was also investigated. The mean serum concentrations of zinc, copper, iron, vitamin A and vitamin E were 0.74 +/- 0.25, 1.18 +/- 0.30, 0.76 +/- 0.20, 0.30 +/- 0.15 and 5.87 +/- 0.43 mg/L, respectively. There was a tendency for SZn to increase with age, whereas SCu and SVA decreased. There was no significant difference in serum trace elements and fat-soluble vitamin concentration between males and females in the different age groups. SFe tended to be lower than that reported in the literature. However, the age groups studied showed no statistically significant sex- and age-related differences. The present study shows that there is a complex interaction between SZn, SCu, SFe, SVA, SVE and age of the children. Multiple regression analysis showed serum zinc was strongly related to serum copper, and serum iron. Serum vitamin A was strongly related to serum zinc and serum vitamin E, whereas serum vitamin E was strongly related to serum zinc, serum copper, and serum vitamin A. On the other hand, our observations also suggest that more detailed studies of these metals and fat-soluble vitamins should be carried out, and that the study should include nutritional surveys, metabolic balances and associations between SZn, SCu, SFe, SVA and SVE and anthropometric variables (height, weight, body mass index and skinfold thickness).

Alterations in kidney enzyme pattern in acute hypervitaminosis A.

The relation of excessive doses of vitamin A with various kidney pathologies is well known however, information concerning the relation of kidney enzyme activity with acute hypervitaminosis A is rather scarce. In this study we describe the kidney enzymatic alterations observed in rats that received daily intramuscular injections of 10,000, 30,000, 50,000 and 100,000 IU of vitamin A palmitate (VA) during seven days (TREATED GROUPS). A comparison is made with the enzyme activity in healthy rats pair-fed and treated with sodium palmitate by intramuscular injection (CONTROL GROUP). The treated rats showed a proportional increase (p < 0.05) in activity of acid maltase, transminases or aminotransferases (GOT and GPT), alkaline phosphatase (ALP) and acid protease with all doses of VA administered. Amylase, lipase and arginase tend to decrease (p < 0.05) in activity only with doses of 50,000 and 100,000 I.U. of VA. Several factors are responsible for these findings, such as kidney necrosis due to release of lysosomal acid hydrolases produced by hypervitaminosis A.

[Effect of cooking on protein digestibility of sorghum (Sorghum bicolor (L.) Moench)]. / Efecto de la cocción sobre la digestibilidad proteica del sorgo (Sorghum bicolor (L.) Moench).

The changes in protein digestibility that occur during cooking have interested many scientists. In this study the effect of cooking sorghum in water on the in vitro protein digestibility (IVPD) was evaluated using sorghum grains not containing tannin (SST), and grains containing detoxified tannin (SPD). The results were compared with rice and maize. The effect of sulfites present in the water used for cooking was also determined. The IVPD of sorghum excent of tannins before cooking was 71.1% smaller (p < 0.05) than that obtained for com (80.8%), polished rice (90.6%) or sorgum with detoxified tannins (80.4%). After cooking in water the IVPD decreased to 23.1%, 66.3%, 3.1% and 3.2% for SST, SPD, polished rice and corn endosperms, respectively. The IVPD of SST and SPD treated with 0.1M sodium bisulfite was 65.2 and 50.1%, which corresponds to a decrease in IVPD of 8.0 and 37.7%, respectively. Similar results were obtained when 0.1M sodium metabisulfite is added to the cooking media. These findings demonstrate that sulfites inhibit the sudden decrease of the IVPD of cooked sorghum grains, and suggest that these compounds may block the formation of disulfide bridges (-S-S-) among the gamma-kafirins molecules located on the surface of the sorghum protein bodies or possibly other factors involved which will be later studied.

Efecto de la cocción sobre la digestibilidad proteica del sorgo (Sorghum bicolor L.) Moench) / Effect of cooking on the protein digestibility of sorghum

Los cambios en la digestibilidad de las proteínas que ocurren durante la cocción han interesado a muchos científicos. Por esta razón en el presente estudio se evaluó el efecto de la cocción en agua sobre la digestibilidad proteca in vitro (DPIV) de granos de sorgo sin taninos (SST) o con taninos previamente detoxificados (SPD) y se comparó con la del arroz y la del maíz. También se estudió la influencia de los sulfitos presentes en el agua de cocción sobre la digestibilidad proteica del sorgo. La DPIV se SST antes de la cocción fue de 71.1 por ciento, menor (p<0.05) que la del SPD, la del maíz y la del arroz pulido, que presentaron respectivamente 80.4 por ciento, 80.8 por ciento y 90.6 por ciento. Todos los granos disminuyeron su digestibilidad después de ser cocidos en agua, la DPIV se redujo en 23.1 por ciento, 66,3 por ciento, 3.1 por ciento y 3.2 por ciento para el SST, el SPD, el arroz pulido y los endospermos de maíz, respectivamente. Los valores de la digestibilidad proteica para el SST y el SPD tratados con bisulfito de sodio (0.1M) fueron de 65.2 y 50.1 por ciento lo cual representa una disminución de la DPIV de 8.0 y 37.7 por ciento, respectivamente. Resultados similares se obtienen con el empleo de metabisulfito de sodio (0.1M) en el medio de cocción. Estos hallazgos demuestran que los sulfitos impiden la disminución brusca de la DPIV de los granos de sorgo sometidos a cocción y sugieren que estos compuestos pueden evitar la formación de los puentes disulfuro entre las moléculas de la gamma-kafirinas (prolaminas) localizadas en la superficie de los cuerpos proteicos del sorgo. Sin embargo, no se descarta la existencia de otros factores que deberán ser estudiados en investigaciones ulteriores. En conclusión, puesto que los sulfitos previenen la formación de puentes disulfuros durante la cocción y hacen al sorgo más digerible por la pepsina, probablemente la formación de los puentes -S-S- es la responsable de la disminución de la digestibilidad proteica del sorgo cocido.

Serum level of Zn, Cu and Fe in healthy schoolchildren residing in Mérida, Venezuela.

Levels of Zn, Cu and Fe were measured in blood serum samples of 320 children: 160 boys and 160 girls randomly selected, ages between 7 and 14 years, all considered healthy and residing in the City of Merida, Venezuela. The metals were determined using flow injection analysis-flame atomic absorption spectrometry. There was a tendency for serum Zn (SZn) to increase with age. There was no significant difference in SZn levels between males and females in the different age groups. Serum copper (SCu) decreases significantly (p < 0.05) with age in male children, whereas it increases in female children. The concentration of serum iron (SFe) tends to be lower than that reported in the literature. However, the age groups studied showed no statistically significant sex and age-related differences. The results are compared with values previously reported for healthy children studied in other communities. The present study has shown that there is a complex interaction between SZn, SCu, SFe and age and sex of the children. On the other hand, our observations also suggest that more detailed studies of these metals should be done, and that the study should include metabolic balances and associations between SZn, SCu, SFe and anthropometric variables (height, weight, body mass index and skinfold thickness).

Tannin elimination and improvement of the digestibility of protein sorghum grains.

Three hybrids of sorghum grains [Sorghum bicolor (L) Moench] containing 3.8, 3.0 and 0.2% of tannins were treated. Abrasive dehulling and storage of moist grains were tested separately and in combination to reduce the tannin content and to improve the nutritional quality of grains. The moisture content of the grains was increased from 12 to 30% by humidifying them with water, acetic acid, sodium bicarbonate or sodium hypochlorite solutions. Abrasive dehulling of the grains to a yield between 75 and 80%, humidifying the grains with acetic acid (1% v/v) and storing them during 7 days at 20 degrees C proved to be the most effective procedure. In this way tannin can be totally reduced and the in vitro digestibility of protein can be increased to 87.5%.

Manganese levels in serum of healthy Venezuelan infants living in Mérida.

Taking up where a previous paper had left off (10) the purpose of this study was to examine in further detail the serum concentration of manganese of 180 apparently healthy Venezuelan infants (96 boys and 84 girls) ranging from 5 days to 12 months old, all residents of Mérida. The flow injection analysis-atomic absorption spectrophotometric technique was used for the determination of manganese. The mean values of serum manganese were 0.42 +/- 0.12, 0.41 +/- 0.11, 0.39 +/- 0.13, 0.39 +/- 0.1, 0.38 +/- 0.09, 0.37 +/- 0.11, 0.36 +/- 0.12 and 0.29 +/- 0.10 microgram/L in infants 5 days and 1,3,5,7,10,11 and 12 months old, respectively. These values indicate that the average concentration of manganese in serum decreases with age, but the mechanism involved is not yet known, nor are the consequences of the decrease. The statistical analysis did not show any significant influence of sex on the serum value of the metal in the age range of 5 days to 12 months.

Effect of isofluorane anesthesia on serum levels of Zn, Cu, Fe, Mn and Co in humans.

In this study the levels of zinc, copper, iron, manganese and cobalt were determined in serum of 25 male and 15 female patients, aged 18 to 35 years, with various pathologies requiring surgery, and who had received isofluorane anesthesia. Significant differences were detected in the concentration of zinc, copper, iron and manganese between presurgical and post-anesthesia samples. The truly striking finding in the present study was the significant increase in serum Zn in the post-operative period. The fact that alterations in the serum levels of these elements occurred only after a considerable time had elapsed following the intervention (24 hours), leads to the conclusion that the changes in serum cation concentration very likely result from surgical trauma and not from isofluorane anesthesia per se.

Halothane anesthesia and serum electrolytes.

The disparate observations on the effect of halothane anesthesia on the serum electrolyte levels in humans prompted us to carry out this work. In this study the levels of sodium, potassium, magnesium, calcium, chloride and inorganic phosphorus were determined in serum of 25 male and 15 female patients, with an age range of 15 to 40 years, who had various pathologies requiring surgery and who were given halothane anesthesia. Significant difference were detected in the concentrations of sodium, potassium, calcium and inorganic phosphorus between presurgical and post-anesthesia induction samples. The truly striking finding in the present study was the significant increase in serum inorganic phosphorus in the intra-operative period. It is suspected that this increase is due to a defect in phosphorylating mechanisms which leads to a rapid hydrolysis of stored and preformed ATP.

Changes of serum lipids in vitamin K3 (menadione) treated rats.

The effect of high doses of vitamin K3 (10 to 50 mg/kg/day of menadione, administered intramuscularly) on the serum content of total lipids, cholesterol, phospholipids and triglycerides in male Wistar rats was evaluated. This experimental group was compared with another group that received intramuscular injections of 10 to 50 mg/kg/day of sodium bisulphite. Hypervitaminosis K3 was diagnosed by jaundice (due mainly to unconjugated bilirrubin) and by anemia which was particularly evident at doses of 40 and 50 mg/kg/day of menadione. These doses of menadione increased serum content of total lipids, phospholipids, and triglycerides but decreased cholesterol. The results show that overdoses of vitamin K3 alter lipid metabolism. The influence o participation of liver damage, fastening, as well as various endocrine and hematological changes are considered responsible for the alterations in serum lipids.

Effects of acute overdose of vitamin A on the hepatic content of K, Na, Mg, Fe, Cu and Zn, in rats.

Interactions among vitamin A metabolism and several metals have been reported in both normal and pathological situations. In the present report we studied, in rats, the effect of daily injections of 100.00 U.I. of vitamin A during seven days on the content of K. Na, Mg, Fe, Cu and Zn in the whole liver. The results were compared with the findings in pair-fed non-treated animals (Control Group). The mean +/- SEM for the concentrations of these elements in the control group were 3.433 +/- 234 (K), 649 +/- 19 (Na), 239 +/- 5 (Mg), 169 +/- 7 (Fe), 5 +/- 0.1 (Cu) and 31 +/- 2 (Zn) ug/g wet tissue, respectively. The hypervitaminosis A, confirmed by a significant increase in tissue concentration of the vitamin, altered the hepatic content of the above mentioned cations. While Na and Zn increased, the other cations decreased its concentrations in the whole liver. Possible mechanisms for these findings are discussed and it is concluded that high doses of vitamin A result in marked changes in the hepatic content of the studied metals.

Concentration changes of electrolytes in serum of chronic chagasic myocardiopathic patients. Sodium, potassium, chloride, calcium, inorganic phosphorus and magnesium.

Sodium, potassium, chloride, calcium, inorganic phosphorus (Pi) and magnesium contents in the serum of 75 myocardiopathic chagasic patients together with that in 29 healthy controls were determined. Blood samples were taken by catheterization from the superior cava vein (SCV), coronary sinus (CS), pulmonary artery (PA) and a peripheral artery, usually the femoral (FA). The results show that the average serum sodium, potassium, chloride, calcium and PI were invariably different in most samples, except the levels of potassium in SCV and Pi levels in FA. The serum concentration of magnesium was significantly different only in the blood of CS. In general one can observe that in the chagasic patients the serum sodium, chloride and calcium tend to diminish while serum potassium, Pi and magnesium tend to increase, especially in CS blood. The coronary gradient indicates that sodium, chloride and calcium tend to deposit in heart tissues, whereas the other elements (potassium, magnesium and Pi) tend to be released from this tissue. The systemic and peripheric gradients suggest that the chemical elements under study tend either to deposit in/or be released from different tissues depending on the course of development of the chagasic myocardiopathy.

[Hematologic changes in rats treated with high doses of vitamin K3 (menadione)]. / Alteraciones hematológicas en ratas tratadas con dosis elevadas de vitamina K3 (menadiona).

The present study describes the effect of high doses of vitamin K3 (8-30 mg/kg) administered daily by intramuscular injection during seven days; on blood smear, red blood cells (RBC) and concentration of hemoglobin (Hb) counts, value of hematocrit (Ht), mean corpuscular hemoglobin concentration (MCHC), reticulocyte and erythroblast counts, total and differential counting of leukocytes (WBC) and total and fractionated serum bilirubin concentration of albino male rats. Results were then compared with findings in normal rats treated with the same doses of sodium bisulfite. In animals treated with vitamin K3 a statistically significant decrease in the number of erythrocytes and in the concentration of Hb, Ht and MCHC was found together with a concomitant increase of erythroblast and reticulocyte counting. In regard to the blood smear, it was observed that erythrocytes vary in their hemoglobin content, size, shape and in their staining properties (polychromatophilia) where as the leukocytes were found to be morphologically normal, but in an increased number. Lymphocytes occasionally showed azurophil granules. Platelets assumed irregular shape and in normal amounts. The leukocytes count showed leukocytosis with marked neutrophilia, eosinophilia, lymphocytosis and monocytosis. Also, the presence of clinical jaundice with an increase of the serum "indirectly-reacting" bilirubin was also observed. These findings indicate that hypervitaminosis K3 induces a marked hemolytic microcytic hypochromic anemia and changes in the white blood cells count. Further studies will be pursued in order to obtain a better understanding of the causes and/or the mechanisms that induce these alterations in the blood cells.

Alteraciones hematológicas en ratas tratadas con dosis elevadas de vitamina K3 (menadiona) / Hematologic changes in rats treatment with high dosis of vitamin K3 (menadione)

En el presente estudio se describe el efecto de dosis elevadas (8 a 30 mg de menadiona/Kg de peso corporal) de vitamina K3 inyectada intramuscularmente, durante un lapso de siete días, sobre el frotis sanguíneo, el contaje de glóbulos rojos, los niveles sanguíneos de hemoglobina (Hb), el valor de hematrocito (Ht), la concentración de la hemoglobina corpuscular media (CHCM), el número de concentración de la bilirrubina total y fraccionada de ratas blancas macho. Luego, se comparan los resultados con lo qeu sucede enratas tratadas con odsis iguales de bisulfito de sodio. En los animales tratados con vitamina K3 se encontró una disminución muy signficativa del recuente de glóbulos rojos, de la Hb, del Ht y de la CHCM, concomitantemente con un marcado incremento en el recuento de eritroblastos y reticulocitos. En el frotis sanguíneo se observó policromasia y variaciones en a forma, tamaño y coloración de los glóbulos rojos; los glóbulos blancos eran morfológicamente normales, aunque aumentados en número; lingocitos ocasionales con gránulos azúrofilos; y plaquetas fusiformes y en cantidades aparentemente normales. En cuanto a la serie blanca,se detectó un incremento significativo en los leucocitos circulantes, con notroia neutrofilia, eosinofilia, linfocitosis y monocitosis. También se notó la presencia d euna ictericia apredominio d ela bilirrubina indirecta. estos hallazgos permiten concluir que la vitamina K en las dosis empleadas produce una marcada anemia hemolítica microcítica hipocrómica, y modificaciones del contaje leucocitario. Estos cambios, por lo tanto, serán objeto de estudios posteriormente, afin de esclarecer las causas y/o los mecanismos que producen esa alteración de las células sanguíneas

Blood serum selenium in the province of Mérida, Venezuela, related to sex, cancer incidence and soil selenium content.

Blood serum selenium levels were measured in healthy subjects of six districts located throughout the province of Mérida, Venezuela. There were either high (87-115 micrograms/L) or low (58-72 micrograms/L) serum selenium-containing districts. Serum selenium in males from three districts were statistically significantly lower (p less than 0.001) than in females. There was a general tendency for serum selenium to increase with age. On the average, a reduction in cancer was observed in the districts with high serum selenium content. Further, serum selenium was lower in male patients from the Mérida district with cancer disease. Association between selenium in soil and in the blood serum of healthy subjects has been observed.

A comparative study of methods for determining selenium in biological materials.

A comparative study of the analytical performance of fluorimetric spectrophotometric, atomic absorption spectrometric, flow injection analysis with atomic absorption spectrometric, flow injection analysis with atomic absorption spectrometric detection, hydride generation with atomic absorption spectrometric detection and hydride generation with molecular emission cavity analysis detection methods has been carried out for the determination of selenium in biological materials. Based on results concerning detection limit, linearity and sensitivity, only the fluorimetric and hydride generation with atomic absorption spectrometric detection methods were suitable for the determination of selenium in biological materials. Whereas, the spectrophotometric, flame absorption spectrometric flow injection-atomic absorption spectrometric and hydride generation with molecular emission cavity detection, due to its worse detection limits and poorer sensitivities, were found to be unsuitable for the determination of selenium in such matrices. The accuracy of the fluorimetric and hydride generation with atomic absorption spectrometric detection methods were tested by using NBS standard reference materials.

A comparative study of methods for determining selenium in biological materials

En el presente estudio se realiza una comparación experimental de las características analíticas de diferentes métodos utilizados para la determinación de selenio en materiales biológicos. Los métodos evaluados fueron: fluorimetría, espectrofotometría de absorción atómica, inyección en flujo continuo con detección por espectrofotometría de absorción atómíca, espectrofotometría y generación de hidruro con detección por espectrofotometría de absorción atómica y emisión molecular en cavidad. Basados en las detecciones límites, lineridades y sensitividades obtenidas para los diferentes métodos, permiten concluir que solamente los métodos de generación de hidruro con detección por espectrofotmetría de absorción atómica y el fluorimétrico pueden utilizarse para la determinación de selenio en materiales biológicos. La exactitud de estos dos métodos se evaluó determinado el contenido de selenio en estándares de la NBS