Supercritical extraction from vinification residues: fatty acids, α-tocopherol, and phenolic compounds in the oil seeds from different varieties of grape.

Supercritical fluid extraction has been widely employed in the extraction of high purity substances. In this study, we used the technology to obtain oil from seeds from a variety of grapes, from vinification residues generated in the Southern region of the state of Rio Grande do Sul, Brazil. This work encompasses three varieties of Vitis vinifera (Moscato Giallo, Merlot, and Cabernet Sauvignon) and two of Vitis labrusca (Bordô e Isabel), harvested in 2005 and 2006. We obtained the highest oil content from Bordô (15.40%) in 2005 and from Merlot (14.66%), 2006. The biggest concentration of palmitic, stearic, and linoleic acids was observed in Bordô, 2005, and in Bordô, Merlot, and Moscato Giallo, 2006. Bordô showed the highest concentration of oleic acid and α-tocopherol in both seasons too. For the equivalent of procyanidins, we did not notice significant difference among the varieties from the 2005 harvest. In 2006, both varieties Isabel and Cabernet Sauvignon showed a value slightly lower than the other varieties. The concentration of total phenolics was higher in Bordô and Cabernet Sauvignon. The presence of these substances is related to several important pharmacological properties and might be an alternative to conventional processes to obtain these bioactives.

Determination of the geographical origin of Brazilian wines by isotope and mineral analysis.

In the present research, we studied wines from three different south Brazilian winemaking regions with the purpose of differentiating them by geographical origin of the grapes. Brazil's wide territory and climate diversity allow grape cultivation and winemaking in many regions of different and unique characteristics. The wine grape cultivation for winemaking concentrates in the South Region, mainly in the Serra Gaúcha, the mountain area of the state of Rio Grande do Sul, which is responsible for 90% of the domestic wine production. However, in recent years, two new production regions have developed: the Campanha, the plains to the south and the Serra do Sudeste, the hills to the southeast of the state. Analysis of isotopic ratios of (18)O/(16)O of wine water, (13)C/(12)C of ethanol, and of minerals were used to characterize wines from different regions. The isotope analysis of δ(18)O of wine water and minerals Mg and Rb were the most efficient to differentiate the regions. By using isotope and mineral analysis, and discrimination analysis, it was possible to classify the wines from south Brazil.

Phenolic content and antioxidant activities of white and purple juices manufactured with organically- or conventionally-produced grapes.

Although the beneficial effects of moderate wine intake are well-known, data on antioxidant capacity of grape juices are scarce and controversial. The purpose of this study was to quantify total polyphenols, anthocyanins, resveratrol, catechin, epicatechin, procyanidins, and ascorbic acid contents in grape juices, and to assess their possible antioxidant activity. Eight Vitis labrusca juices--white or purple, from organically- or conventionally-grown grapes, and obtained in pilot or commercial scale--were used. Organic grape juices showed statistically different (p<0.05) higher values of total polyphenols and resveratrol as compared conventional grape juices. Purple juices presented higher total polyphenol content and in vitro antioxidant activity as compared to white juices, and this activity was positively correlated (r=0.680; p<0.01) with total polyphenol content. These results indicate that white and purple grape juices can be used as antioxidants and nutritional sources.

Elevated gamma-aminobutyric acid, glutamate, and vascular endothelial growth factor levels in the vitreous of patients with proliferative diabetic retinopathy.

OBJECTIVE: To examine the relative levels of gamma-aminobutyric acid (GABA), glutamate, and vascular endothelial growth factor (VEGF) in the vitreous of nondiabetic and diabetic patients. METHODS: Undiluted vitreous samples were obtained from 22 patients with proliferative diabetic retinopathy (PDR) and 28 patients without diabetes who underwent pars plana vitrectomy. Simultaneous venous blood samples also were obtained. Amino acid concentrations were determined using sensitive high-performance liquid chromatography, and VEGF levels by quantitative enzyme-linked immunosorbent assay. Hemoglobin concentrations in the blood and vitreous were determined using spectrophotometry. RESULTS: The level of GABA in the vitreous of patients with PDR, 29.4 +/- 7.8 mumol/L, was significantly higher than in controls (18.4 +/- 5.5 mumol/L) (P = .004). The vitreous concentration of glutamate was higher in patients with PDR (24.7 +/- 14.0 mumol/L) compared with controls (9.1 +/- 5.1 mumol/L) (P < .001). Vitreous VEGF level was significantly higher in patients with PDR (1759 +/- 1721 pg/mL) compared with controls (27 +/- 65 pg/mL) (P < .001). There were moderately strong correlations between GABA and VEGF levels (r = 0.68) and glutamate and VEGF levels (r = 0.43). Elevated GABA, glutamate, and VEGF levels also correlated strongly with the presence of PDR. Correcting for possible introduction of these molecules by vitreous hemorrhage did not significantly alter these findings. CONCLUSIONS: Levels of glutamate potentially toxic to retinal ganglion cells are found in the vitreous of patients with PDR. Elevated vitreous GABA may reflect amacrine cell dysfunction and underlie electroretinographic oscillatory potential abnormalities seen in diabetic retinopathy. The correlations of glutamate and GABA levels with an elevated VEGF level provide biochemical support for ischemia-induced neovascularization in patients with PDR. These findings present opportunities for novel therapeutic modalities in the treatment of PDR.

Linearity and accuracy of ultraviolet and visible wavelength photometer: an interlaboratory survey.

A survey was made to determine the linearity and accuracy of ultraviolet and visible wavelength photometers used by laboratories in New York State. Two solutions each of high-purity potassium dichromate and cobalt ammonium sulfate were submitted for photometric performance studies. The majority of the participant spectrophotometer results showed good correlation with reference data. Broad half-band width (greater than 10 nm) photometers showed little deviation from linearity. Coefficients of variation for the models surveyed were 5-10%.

The effect of temperature on the kinetic constants of human lactate dehydrogenase 1 and 5.

The kinetic constants of human lactate dehydrogenase 1 and 5 (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27), assayed lactate-to-pyruvate increase with temperature. The reaction mechanism is ordered sequential as has been found with lactate dehydrogenase from other sources. The KM values for each substrate are larger for isoenzyme 5 than for 1. For lactate dehydrogenase 1 the KM(lactate) increases from 1.07 mM at 25 degrees C to 3.95 mM at 37 degrees C and for lactate dehydrogenase 5 it increases from 5.37 mM at 25 degrees C to 6.88 mM at 37 degrees C. The KM(NAD+) for lactate dehydrogenase 5 is 0.14 mM at 25 degrees C and 0.29 mM at 37 degrees C. The increase in the KM for each substrate with increasing temperature confirms that additional substrate is required for optimal reaction conditions at higher temperatures.

The National Reference System for Cholesterol.

The NRS/CHOL is a reality that is progressively becoming the way in which accuracy of cholesterol results is being ensured. Proof of traceability to NRS/CHOL within each segment of the clinical laboratory, now an expectation, will soon be the norm that makes a sound conceptual reference system into an everyday reality that reaches into the many thousands of working sites that measure cholesterol in serum on patient samples.

Review of pyridoxal phosphate and the transaminases in liver disease.

In vitro supplementation with the active form of vitamin B6, pyridoxal-phosphate (PLP), increases measurements of both serum aminotransferase enzymes, L-aspartate: 2-oxoglutarate amino transferase, EC 2.6.1.1 (AST) and L-alanine: 2-oxoglutarate aminotransferase, EC 2.6.1.2 (ALT). The plasma PLP level in normal individuals clearly relates inversely to the degree of stimulation of serum AST and ALT. PLP added in vitro increases the reference values but does not decrease the biological variability of AST measurements in healthy individuals. Since B6 deficiency is observed in alcoholics, in some significant percentage of hospitalized patients and in apparently healthy people over age 64, these individuals will show PLP stimulation of their serum amino-transferase enzymes. Patients with liver disease show lesser activation with PLP of AST activity but not ALT activity than patients with heart disease (myocardial infarction). AST isoenzyme measurements in the form of a mitochondrial AST/total AST ratio may discriminate alcoholic hepatitis from all other hepatic diseases. In renal dialysis patients including transplant patients, it may be desirable to measure the aminotransferases with added PLP in order to reflect better the cytolytic state of the liver. While unconfirmed studies suggest the combination of PLP activation and AST isoenzyme measurements may aid in the diagnosis of hepatoma, PLP activation per se does not provide clear cut improved diagnostic value of AST and ALT in liver diseases. However, in view of PLP incorporation into the IFCC reference methods for AST and ALT, and the National Reference System for the Clinical Laboratory, it is recommended that PLP be included in all AST and ALT measurements.

Urinary enzyme measurements in the diagnosis of renal disorders.

In view of Mattenheimer's comprehensive review of enzymes in renal disease in this journal only four years ago, major emphasis has been placed at the recent Symposium on Diagnostic Significance of Enzymes and Proteins in Urine held at Kanderstag, Switzerland in March of 1978. The pathophysiology of enzyme release in renal disease is presented, the current status of enzyme measurement reviewed, and the laboratory findings in several main types of renal disease summarized. While the measurement of selected urinary enzymes has been found extremely useful in specific situations, most investigators agree that routine screening is not warranted at this time. Newer developments in the measurement of isoenzyme patterns hold promise of introducing increased diagnostic specificity and appear to be the wave of the future.

Measurement of total lactate dehydrogenase activity.

Lactate dehydrogenase (LD: EC 1.1.1.27) is the most important clinically of several dehydrogenases occurring in human serum. Lactate dehydrogenase is cytoplasmic in its cellular location and in any one tissue is composed of one or two of five possible isoenzymes. While many of its clinical applications involve quantification of one or more specific serum isoenzymes, an estimate of total LD is required usually. Lactate dehydrogenase catalyzes the reversible reaction: L-lactate + NAD+ in equilibrium pyruvate + NADH. The bidirectional reaction is monitored spectrophotometrically by measuring either the increase in NADH at 340 nm produced in the lactate-to-pyruvate reaction (L----P) or by the decrease in NADH at 340 nm produced in the pyruvate-to-lactate (P----L) reaction. Kinetic assay systems for the measurement of the reaction system in both directions are comprehensively reviewed as well as the standardization efforts proposed to date.

Survey of special practices associated with College of American Pathologists proficiency testing in the Commonwealth of Pennsylvania.

A questionnaire concerned with special practices associated with proficiency testing was sent to the Chemistry Supervisor, Hematology Supervisor, and Chief Pathologist of 190 Pennsylvania hospital laboratories. Responses were received from 156 hospitals (82.1%) and included responses from 131 chemistry supervisors, 108 hematology supervisors, and 92 chief pathologists. The vast majority of respondents (85% to 95%) indicated moderate to great pressure to score acceptably. The survey showed a high prevalence of special practices, including analysis of controls just prior to survey specimens (23% to 42%), analysis in duplicate on a single instrument (52% to 88%), analysis on more than one instrument (17% to 31%), analysis on two or more separate days (20% to 39%), and delay of testing until an instrument is "working better" (24% to 34%). Approximately 63% of chemistry results and 72% of hematology results are reported as averages or medians. Pathologists consistently reported a lower prevalence of special practices than did laboratory supervisors. These high prevalences of special practices associated with proficiency testing specimens have important implications for proficiency testing programs.

Phenolic composition and antioxidant activity in sparkling wines: modulation by the ageing on lees.

Sparkling wines (SW) have a special biological ageing on lees that is performed using two distinct methods: in the bottle (Champenoise) or in isobaric tanks (Charmat method). The objective of this study was to compare the levels of phenolic compounds, ß-Glucosidase and antioxidant activity during the ageing on lees, in samples of SW produced at industrial scale by both methods. The ß-Glucosidase activity has been constant over time, showing a close relationship with all the polyphenols studied (resveratrol, piceid, tyrosol, gallic, caffeic and ferulic acids), which were affected by the sur lie time. With these cross-reactions, the biological properties of the SW were also modulated. The results showed that the long period of ageing decreased the antioxidant potential in all samples. This work demonstrates that the sur lie is more important than the production method itself, due to its ability to modulate the necessary changes to achieve the specific objective.

Characterization of wines according the geographical origin by analysis of isotopes and minerals and the influence of harvest on the isotope values.

We studied Brazilian wines produced by microvinification from Cabernet Sauvignon and Merlot grapes, vintages 2007 and 2008, from the Serra Gaúcha, Campanha and Serra do Sudeste regions, in order to differentiate them according to geographical origin by using isotope and mineral element analyses. In addition, the influence of vintage production in isotope values was verified. Isotope analysis was performed by isotope ratio mass spectrometry (IRMS), and the determination of minerals was by flame atomic absorption (FAA). The best parameters to classify the wines in the 2008 vintage were Rb and Li. The results of the δ(13)C of wine ethanol, Rb and Li showed a significant difference between the varieties regardless of the region studied. The δ(18)O values of water and δ(13)C of ethanol showed significant differences, regardless of the variety. Discriminant analysis of isotope and minerals values allowed to classify approximately 80% of the wines from the three regions studied.

Effects of buffers on aspartate aminotransferase activity and association of the enzyme with pyridoxal phosphate.

Using purified enzymes of human origin and patients' sera, we examined factors influencing the in vitro association of pyridoxal phosphate with aspartate aminotransferase (EC 2.6.1.1). The rate of association was markedly retarded by phosphate buffer in comparison with tris(hydroxymethyl)aminomethane or six other buffers. Pyridoxal phosphate at an incubation concentration of 130 mumol/liter reactivated the entire apoenzyme portion of an apoenzyme/holoenzyme mixture within 5 min in tris(hydroxymethyl)aminomethane; in contrast, less than 20% was associated during 15 min in phosphate. Activity measured in tris(hydroxymethyl)aminomethane-buffer without exogenous pyridoxal phosphate was 4% greater than that in phosphate and was slightly increased by increasing the pH of the assay mixture from 7.5 to 8.0. Aspartate in the incubation medium did not retard the stimulation in tris(hydroxymethyl)aminomethane buffer. While the magnitude of stimulation varied greatly among sera, a consistent mean stimulation of 30% for groups of sera with normal activities was found when asparate at 125 mmol/liter, 2-oxoglutarate at 6.7 mmol/liter and tris(hydroxymethyl)aminomethane at 90 mmol/liter were used, an increase over the 16% with phosphate buffer [Clin. Chem. 19, 92 (1973)]. Absorbance spectra suggest pyridoxal phosphate exists as the Schiff base of tris(hydroxymethyl)aminomethane or aspartate, or both, under conditions of assay incubation (without addition of 2-oxoglutarate). Nonenzymatic catalysis of the reaction by pyridoxal phosphate alone or a formation of a protein/pyridoxal phosphate adduct was discounted with use of a D-asparate substrates.

Effects of temperature on the steady-state kinetics and measurement of aspartate aminotransferases.

We examined the effects of temperature on the activity and steady-state kinetics of aspartate aminotransferase (EC 2.6.1.1), using purified human soluble (s-AspAT) and mitochondrial (m-AspAT) isoenzymes, human serum, and porcine s-AspAT. All enzymes obeyed similar linear Arrhenius relationships over the range 20-40 degrees C. Apparent energies of activation (52.3 kJ.mol-1) and ratios of activity between 30 and 37 degrees C (0.626) were identical for the human s- and m-AspAT. This ratio was 0.623 (SEM 0.004) for human sera; deviation from the predicted ratio by individual sera was within analytical error. Similar activity/temperature relationships were observed for porcine s-AspAT. The use of factors to convert AspAT activities at 30 and 37 degrees C influenced neither precision of measurement of frequency distributions of results. The apparent Michaelis constants for the human isoenzymes increased with temperature. The least-influenced Km was for 2-oxoglutarate and s-AspAT: K2-oxoglutarate was 0.24 mmol.L-1 at 25 degrees C and 0.29 mmol.L-1 at 37 degrees C; apparent enthalpy change for substrate binding (delta HS) was 12.1 kJ.mol-1. The largest variation was for 2-oxoglutarate and m-AspAT: K2-oxoglutarate was 0.46 mmol.L-1 at 25 degrees C and 1.02 mmol.L-1 at 37 degrees C; delta HS was 50.8 kJ.mol-1. Incubation of the human isoenzymes with substrate mixture (without 2-oxoglutarate) at 23 and 37 degrees C did not affect activity during 60 min if tris(hydroxymethyl)aminomethane buffer was used. When the isoenzymes were diluted to 10 nmol-L-1 (about 200 U.L-1) in buffer alone and incubated at 50 degrees C, m-AspAT activity was decreased by 20% after 120 min; the cytoplasmic enzyme was unaffected.

Quantitative toxicology: interlaboratory and intermethod evaluation in New York State.

The New York State Department of Health has conducted a proficiency evaluation program in quantitative toxicology since 1974. Serum samples containing a barbiturate and phenytoin, together with either glutethimide, procainamide, or theophylline, are sent to participating laboratories quarterly. Within the first two years of the program the percentage of laboratories able to quantitate 75% of the test samples to within 25% of the gravimetric values increased from 25 (1974-1975) to 40% (1975-1976). This improvement was partly due to licensure requirements, improved technology for sample preparation and analysis, and the availability of better quality-control practice. An obstacle to obtaining uniform accuracy is the lack of adequate calibration or testing materials. To overcome these obstacles, pure drugs are weighed into a bovine serum matrix, and the weights are confirmed by reference laboratories and used as the target values in the testing program. Comparison of the methods used by participants in this program for barbiturate and phenytoin yielded equations different from those found in other method evaluations.

Interlaboratory proficiency, intermethod comparison, and calibrator suitability in assay of serum aspartate aminotransferase activity.

Sources of variation in assays of aspartate aminotransferase (EC 2.6.1.1) activity were examined in an interlaboratory survey and through an examination of materials used as calibration materials in these assays. Four highly stable lyophilized specimens containing human cytoplasmic enzyme, with activities of 0, 22, 46, and 96 U/liter at 30 degrees C and optimal substrate concentrations, were assayed by 319 laboratories. Mean values obtained on these specimens by laboratories using 2,4-dinitrophenylhydrazine kits varied among manufacturers and deviated from values expected from this procedure. The average coefficient of variation (CV) with these kits was greater than 20%. Automated continuous-flow procedures with use of diazonium salt showed the best precision (av CV, less than 10%). However, the automated continuous-flow malate dehydrogenase/NADH coupled method produced an average CV greater than 20%. Results from each of the automated methods were related to a reference malate dehydrogenase/NADH coupled continuous kinetic assay method by temperature relationships alone. Mean values from manual diazonium salt procedures were 1.7-fold greater than similar reference values (av CV was 18%). The higher results were attributed to the use of poorly-defined units and to an artifact caused by chromophore stabilizers in this procedure when aqueous samples are used. The average CV in continuous kinetic methods varied among kit manufacturers, ranging from 6 to 28% for the specimen of highest activity. Variations in results were much larger at 366 nm than at 340 nm than at 340ity. Variations in results were much larger at 366 nm than at 340 nm. Interassay relationships of these methods are presented. Concentrations of pyruvate in commercially available calibration materials differed between manufacturers, varied in stability, and deviated from the expected concentration. For some colorimetric assays the precision attained on reported absorbance values for the enzyme specimens was of the same order of magnitude as that for pyruvate standards. Other sources of error are revealed by the interlaboratory survey. The value of commercially available sources of enzyme activity as calibration or control materials was assessed by evaluating the following properties: activity at suboptimal concentrations of L-aspartate or 2-oxoglutarate, temperature effects, preincubation lability owing to aspartate and phosphate, pyridoxal phosphate saturation, contamination with glutamate dehydrogenase, and manufacturer's rated activity. These properties are compared to those of human cytoplasmic enzyme in a human serum matrix.