Commercial Enzymatic Kit Research Articles

Effects of Quercetin Nanoemulsion on Cholesterol Efflux and MicroRNA-33/34a Expression in the Liver of Mice Fed with a High-Cholesterol Diet.

Quercetin is a flavonoid widely present in plants; despite its beneficial physiological activity, it exhibits considerably low bioavailability. Nanoemulsion technology is used for improving the bioavailability of lipophilic phenolic compounds. This study aimed to investigate the potential effects of quercetin nanoemulsion (QN) on regulating the microRNA (miR)-33/34a pathway involved in cholesterol efflux in the liver of mice fed with a high-cholesterol (HC) diet. Subsequently, C57BL/6J mice were divided into four groups and fed a normal chow diet, HC diet supplemented with 1% cholesterol and 0.5% cholic acid, or HC diet supplemented with 0.05% QN or 0.1% QN for 6 weeks. Serum and hepatic lipid profiles were assayed using commercial enzymatic kits. Gene expression and miR levels were quantified using real-time quantitative reverse transcription polymerase chain reaction, and adenosine monophosphate-activated protein kinase (AMPK) activity was measured using an AMPK Kinase Assay kit. QN supplementation improved serum and liver lipid profiles. QN upregulated the mRNA levels of adenosine triphosphate (ATP)-binding cassette subfamily A1, ATP-binding cassette subfamily G1, and scavenger receptor class B type 1, which are related to cholesterol efflux. In the QN group, the hepatic AMPK activity increased, whereas miR-33, and miR-34a expression levels decreased. These results suggest that QN may enhance cholesterol efflux, at least partly through modulating AMPK activity and miR-33/34a expression in the liver.

New enzymatic method for estimating fumaric acid in wines

This work aims to develop a new enzymatic method for analysing fumaric acid in wines. Fumaric acid is a very effective lactic acid bacteria inhibitor that is widely used in the food industry as an additive (E297). Using it to inhibit malolactic fermentation has recently been authorized by the International Organisation of Vine and Wine (OIV). However, the official analytical method for fumaric acid involves the classical HPLC method for organic acids, which is overly complicated for use in wineries. This research proposes a very simple and efficient enzymatic method that allows the simultaneous determination of L-malic acid and fumaric acid. The method uses a commercial enzymatic kit for L-malic acid and adds a supplementary step in which the fumarase enzyme is added to transform fumaric acid into L-malic acid. The results obtained show that this method could be used in different media (synthetic solution, white wine, red wine and white grape juice). This article also proposes a modification of the enzymatic method to be applied when the L-malic acid concentration of the sample is very high.

Ipomea batatas Leaf Powder from Cameroon: Antioxidant Activity and Antihyperlipidemic Effect in Rats Fed with a High-Fat Diet.

The present study consists of analyzing the phytochemical composition of Ipomoea batatas leaf powders and evaluating their antihyperlipidemic effect on rats receiving a high-fat diet. Ipomoea batatas leaves were collected from four agroecological areas of Cameroon, and powders were obtained after washing, drying, grinding, and sieving. Standard analytical methods were used to determine the phytochemical composition of two varieties (IRAD-tib1yellow-V1 and IRAD-1112white-V2) from North Z1, Adamawa Z2, West Z3, Center Z4. The effect of I. batatas leaf powder on lipid metabolism was assessed in vivo by feeding different groups of rats with a high-fat diet supplemented with 5 and 10% of I. batatas leaf powder during 30 days. At the end of the experimentation, total cholesterols, triglycerides, LDL- (low-density lipoprotein-) cholesterol, HDL- (High-density lipoprotein-) cholesterol, ASAT (aspartate aminotransferase), ALAT (alanine aminotransferase), and creatinine were measured using commercial enzymatic kits (Spinreact, Spain). The results of phytochemical analysis of I. batatas leaf powders revealed that the total phenol content ranged from 660.173 mg GAE/100 gDW (Z1V2) to 657.76 mg GAE/100 gDW (Z3V2), the flavonoids content ranged from 282.25 mgEC/100 gDW (Z3V1) to 325.05 mgEC/100 gDW (Z4V2), and the anthraquinone content ranged from 324.05 mg/100 gDW (Z3V2) to 326.72 mg/100 gDW (Z4V2). The total antioxidant capacity ranged from 19.00 (Z1V1) to 23.48 mg AAE/gDW (Z3V2), while the IC50 ranged from 1.58 mg/mL (Z1V1) to 3.08 mg/mL (Z3V2). Rats fed a high-fat diet and supplemented with 5 and 10% of I. batatas leaf powder showed a significant (p < 0.05) reduction in body weight compared to the control with a reduction rate ranging from 6 to 10%. The consumption of I. batatas leaf powder for 30 days significantly (p < 0.05) reduced serum total cholesterol, LDL-cholesterol, triglycerides, ALAT, and creatinine level. These results suggest the use of I. batatas leaves as a phytomedicine in the treatment of cardiovascular diseases.

Paper-Based Enzyme Biosensor for One-Step Detection of Hypoxanthine in Fresh and Degraded Fish.

Food freshness monitoring, which can reflect the quality of the product at the time of use, remains a great challenge for consumers and the food industry. Herein, we report the development of a cost-effective enzyme-based paper biosensor, which can monitor fish freshness and predict spoilage. The biosensor measures the release of hypoxanthine (HX), an indicator of meat and fish degradation, using the enzymatic conversion of HX by xanthine oxidase (XOD). We demonstrate that the entrapment of XOD and an organic dye, nitro blue tetrazolium chloride (NBT), within a sol-gel biohybrid enables their stabilization on paper and promotes the enzymatic reaction with further retention of the reaction products within the cellulosic network . Linearity in the micromolar concentration range with a detection limit of 3.7 μM for HX is obtained. The biosensor has high selectivity toward HX and is manufactured in few steps from inexpensive widely available materials. The applicability of the biosensor is demonstrated by following fish degradation over time and measuring HX concentrations ranging from 117 (±9) to 198 (±5) μM within 24 h of degradation, at levels that are comparable with those measured by a commercial enzymatic kit for HX detection. As compared to the commercial kit, our biosensors are more cost-effective, do not require addition of exogenous reagents and are portable, having all of the reagents needed for analysis embedded within the sensing platform. This proof-of-concept work demonstrates that the paper-based HX biosensor has potential as a robust reagentless device for real-time monitoring of food freshness and for other applications in which HX plays an important role.

Technical note: Evaluation of 3 methods to determine mucin protein concentration in ileal digesta of young preweaning calves

The use of alternative sources of protein to substitute for milk proteins in milk replacers (MR) can increase the synthesis of endogenous proteins and therefore alter ileal or total-tract digestibility calculations. Mucin is the main component of gastrointestinal mucus and represents the greatest contribution to total endogenous protein. Mucin is difficult to isolate and has not been extensively studied in dairy calves. We explored 3 different procedures to analyze and estimate mucin protein (MUP) in ileal digesta of young dairy calves. Ileal digesta samples were collected from nine 30-d-old ileal-cannulated calves that were enrolled in a 3 × 3 replicated Latin square with 5-d periods. The 3 diets were a control whey protein-based MR (WPC), an isonitrogenous MR in which 50% of the protein was from enzyme-treated soybean meal (ESBM), and an N-free MR (NFREE). Mucin protein concentration and flow were analyzed by fractionation of the digesta and ethanol precipitation; this process served as the reference method. Alternative methods to estimate MUP consisted of using commercial enzymatic kits to analyze glucosamine (N-acetylglucosamine, GlcNAc) and galactosamine (N-acetylgalactosamine, GalNAc), 2 amino-sugars that are highly enriched in mucin. Before GlcNAc determination, samples were processed using 3 different procedures: sample clarification (GLCL), clarification plus hydrolysis (GLCH), and hydrolysis alone (GLHL). The MUP was estimated by regression of the GlcNAc and GalNAc values using previously validated equations. According with the bias and agreement analysis, none of the methods yielded MUP values similar to the reference method. However, GLHL showed a strong association with the reference method (ρ = 0.73). It allowed identifying the smaller MUP flows with NFREE compared with the other 2 diets and detecting the greater flow of ESBM than WPC, as observed with the reference method. Using the GlcNAc values from GLHL and the MUP measured with the reference method, we were able to establish a linear relationship between both methods (adjusted R2 = 0.75). We found that the GLHL method enabled detecting differences in MUP ileal flows between diets differing in protein level and source. Inferences about MUP secretions must be done cautiously because many dietary and physiological factors are involved. The adoption of practical techniques to determine MUP can help to increase our knowledge about gastrointestinal tract function and to improve the accuracy of MR digestibility calculations.

Icariin improves acute kidney injury and proteinuria in a rat model of pregnancy‑induced hypertension.

Icariin has previously been demonstrated to attenuate hyperglycemia-induced renal injury, however the renoprotective effects of icariin in a rat model of pregnancy-induced hypertension (PIH) remain to be elucidated. The present study aimed to investigate the effect of icariin on PIH-induced acute kidney injury (AKI) and proteinuria. Following 18 days of icariin treatment between day 1 and day 18 of gestation, which was combined with NG-nitro-L-arginine methyl ester (L-NAME) treatment between day 12 and day 18 of gestation to induce PIH, the 24 h urine protein level, blood urea nitrogen and serum creatinine were measured by using the Coomassie Brilliant Blue method, a commercial enzymatic kit and the picric acid method, respectively. Renal tissues were collected at day 18 of gestation for hematoxylin and eosin staining and immunohistochemistry. The mRNA expression of AGT and protein expression of angiotensin II (Ang II) in the kidneys of control and PIH rats was investigated by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively, to determine the effect of icariin on components of the renin-angiotensin system. The results demonstrated that L-NAME treatment in pregnant rats resulted in significant increases in systolic blood pressure (SBP) and diastolic blood pressure, in addition to the induction of severe proteinuria. The significant increase in SBP and proteinuria in PIH rats was prevented by icariin. L-NAME-induced AKI resulted in profound renal histological alterations, including mesangial expansion and glomerular lesions. L-NAME administration exerted a marked decrease in the mRNA and protein expression levels of nephrin in the kidneys from PIH rats compared with control group. Furthermore, upregulation of circulating and renal Ang II levels in PIH rats was observed. However, icariin treatment significantly reversed the L-NAME-induced downregulation of nephrin and upregulation of circulating and renal Ang II levels in PIH rats. These results demonstrated that icariin administration improved urinary protein excretion levels and renal tissue damage in PIH rats, and the underlying mechanism was mediated in part, via upregulation of nephrin expression and downregulation of Ang II.

Heme oxygenase-1 (HO-1) protects human lens epithelial cells (SRA01/04) against hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis

ObjectivesThis study aimed to investigate the protective role of heme oxygenase-1 (HO-1) in H2O2-induced oxidative stress and apoptosis in human lens epithelial cells (hLEC; SRA01/04). MethodsSRA01/04 cells were exposed to a hydrogen peroxide (H2O2) concentration gradient and inducers of HO-1 such as cobalt protoporphyrin (CoPP) and zinc protoporphyrin (ZnPP), respectively. In addition, an RNA silencing experiment was conducted to investigate the HO-1 function in this study. A Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability. Western blot and ELISA were used to detect the level of HO-1 expression. In our study, hLECs were exposed to 400 μM hydrogen peroxide (H2O2) for 24 h with or without pretreatment with 10μΜ CoPP or 10μΜ ZnPP, respectively. Double immunofluorescence staining was used for cell identification and the qualitative expression of HO-1. Expression of HO-1 was monitored using Western blot and ELISA. Intracellular reactive oxygen species (ROS) were detected by flow cytometry analyses; commercial enzymatic kits were used to measure the levels of glutathione (GSH), as well as superoxide dismutase (SOD). The proportion of cell apoptosis was quantified by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The expression of caspase family (−8, -3) proteins was measured by Western blot analysis. ResultsHO-1 significantly restored the cell viability under H2O2 injury via reducing the generation of ROS and increasing the levels of SOD and GSH activity. Moreover, HO-1 also inhibited H2O2-induced caspase-8 and caspase-3 proteins, thus significantly reducing the apoptosis of SRA01/04. An RNA silencing experiment demonstrated the increased resistance of LECs to oxidative stress specifically for increased levels of HO-1. ConclusionsThese findings suggested that HO-1 protects human lens epithelial cells from H2O2-induced oxidant stress by upregulating antioxidant enzyme activity, reducing ROS generation, and thus inhibiting caspase family-dependent apoptosis.

Isoflavones and phytosterols contained in Xuezhikang capsules modulate cholesterol homeostasis in high-fat diet mice.

Xuezhikang (XZK), an extract of red yeast rice, has been widely used in traditional Chinese medicine to treat cardiovascular disease. Three fractions F1, F2 and F3 (primarily containing isoflavones, monacolins or phytosterols, respectively) are extracted from Xuezhikang capsules. In this study we evaluated the lipid-lowering effects of these fractions and explored the potential mechanisms of actions. Mice treated with a high-fat diet (HFD) were orally administered lovastatin (10 mg·kg(-1)·d(-1)), XZK (1200 mg·kg(-1)·d(-1)), F1 (27.5 mg·kg(-1)·d(-1)), F2 (11.3 mg·kg(-1)·d(-1)) or F3 (35 mg·kg(-1)·d(-1)) for 10 weeks. Lipids were measured using commercial enzymatic kits, and the mRNA and protein levels of genes involved in cholesterol and bile acid homeostasis were evaluated using qRT-PCR and Western blot analysis, respectively. XZK increased the fecal excretion of lipids and bile acids, reduced serum TC, TG and LDL-C levels by 40%, 55% and 46%, respectively, and increased serum HDL-C by 31%. Administration of F1 repressed serum TC and TG by 24% and 52%, respectively, and elevated hepatic synthesis of CYP7A1. It also increased hepatic elimination of bile acids in the fecal excretions by 79% through upregulating BSEP and downregulating NTCP. Administration of F3 decreased serum TC, TG and LDL-C levels by 33%, 29% and 39%, respectively, and increased serum HDL-C by 28%, significantly reduced intestinal absorption of cholesterol by inhibiting the transcription of NPC1L1, and elevated excretion of TC, FC and CE by 96%, 72% and 101%, respectively. Administration of F2 showed pharmacological effects similar to those of lovastatin. Isoflavones and phytosterols in XZK exert cholesterol-lowering effects in HFD mice through mechanisms that differ from those of lovastatin. Isoflavones and phytosterols act in a complimentary manner: through enhancing the elimination of bile acids and reducing intestinal cholesterol absorption, respectively.

Evaluation of Anti-Atherosclerotic Activity of Virgin Coconut Oil in Male Wistar Rats Against High Lipid and High Carbohydrate Diet Induced Atherosclerosis

The aim of this study was to analyze the effect of virgin coconut oil (VCO) on high lipid diet (HLD) and high carbohydrate diet (HCD) induced atherosclerosis in male Wistar rats. Spectrophotometer was used to determine the lipid parameters by enzymatic endpoint method. The plasma total cholesterol (TC), triglyceride (TG) and high-density lipoprotein (HDL) levels were measured using commercial enzymatic kits. The results showed that feeding with normal and VCO diet significantly decrease (p&lt;0.05) body weight when compared to the control group rats. Body weight and lipid profile were estimated after 8 weeks of treatment. VCO showed a significant (p&lt;0.05) reduction in TC, TG, Low density lipoprotein (LDL), Very low density lipoprotein (VLDL) levels and significant (p&lt;0.05) increase in HDL in HCD /HLD group rats. There was significant decrease in atherogenic index (AI) (p&lt;0.05) of all VCO treated groups when compared to the Control group. Hence, there was increase in the percentage of protection in VCO treated animals after 8 weeks.The decrease of lipid profiles in the experimental rats showed that VCO possesses anti-atherosclerotic activity.

Lycium barbarum polysaccharides protect human lens epithelial cells against oxidative stress-induced apoptosis and senescence.

ObjectivesWe aimed to investigate the protective effect of Lycium barbarum polysaccharides (LBPs) against oxidative stress–induced apoptosis and senescence in human lens epithelial cells.MethodsTo study apoptosis, SRA01/04 cells, a human lens epithelial cell lines, were exposed to 200 µM hydrogen peroxide (H2O2) for 24 h with or without pretreatment with LBPs. Cell viability was measured using a Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis, intracellular reactive oxygen species (ROS), and the loss of mitochondria membrane potential (Δψm) were detected by flow cytometric analyses. Expression levels of Bcl-2 and Bax proteins were measured by western blot analysis. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) were quantized using commercial enzymatic kits according to the manufacturer's instructions. To study senescence, SRA01/04 cells were pre-incubated with LBPs and all cells were then exposed to 100 µM H2O2 for 96 h. Cellular senescence was assessed by morphologic examination and senescence-associated β-galactosidase (SA-β-gal) staining.ResultsLBPs significantly reduced H2O2-induced cell apoptosis, the generation of ROS, the loss of Δψm, and the levels of MDA. LBPs also inhibited H2O2-induced downregulated Bcl-2 and upregulated Bax proteins and increased the levels of SOD and GSH enzyme activity. Moreover, LBPs significantly attenuated H2O2-induced cellular senescence.ConclusionsThese findings suggested that LBPs protect human lens epithelial cells from H2O2-induced apoptosis by modulating the generation of ROS, loss of Δψm, Bcl-2 family, and antioxidant enzyme activity and attenuating cellular senescence.

Differentiation between fresh and thawed chicken meats

Adulteration of fresh meat and its possible substitution with frozen and thawed meat represents a problem, since thawed meat has not only lower sensory qualities than fresh meat but also a lower price. Commercial enzymatic kits seem to be a solution to reveal such unfair practises and were applied to detect the activity of specific enzymes, citrate synthase, mitochondrial enzymes that are released from the organelles destroyed by frost. We determined, whether the meat of slaughtered chicken was fresh or frozen/thawed, and to provide convincing results. The absolute results vary with the type of meat and depend on the enzyme used. However, the enzyme activity in the exudates of frozen/thawed meat is always higher than in fresh meat. This value further increases with each subsequent freezing cycle. The determination of citrate synthase activity was done only in the exudate released from the examined meat samples. However, to determine the enzymes activity directly in unpacked meat, which have not released any exudate, is the subject of further research. &amp;nbsp;

Amperometric carbon paste enzyme electrodes for uric acid determination with different mediators

In this study, two new amperometric carbon paste enzyme electrodes for determination of uric acid were developed. The carbon paste was prepared by mixing uricase enzyme, 1,4-benzoquinone or poly(vinylferrocene) (PVF) as a mediator, graphite powder, paraffin oil and then the paste was placed into cavity of a teflon electrode body. Determination of uric acid was performed by oxidation of enzymatically generated H2O2. The effects of enzyme loading, mediator amount, buffer type, pH, buffer concentration, working potential and temperature were investigated for both electrodes. The working range of the 1,4-benzoquinone modified enzyme electrode was 1.9 × 10–8–2.7 × 10–3 M, detection limit 1.9 × 10–8 M and response time 150 s. Optimum buffer type, pH, buffer concentration, working potential, temperature and amounts of enzyme and mediator for 1,4-benzoquinone modified enzyme electrode were found to be Tris, 8.0, 0.20 M, +0.25 V, 30 °C, 2.0 Unit and 13%, respectively. The working range of the PVF modified enzyme electrode was 7.4 × 10–8–7.0 × 10–3 M, detection limit 7.4 × 10–8 M and response time 120 s. Optimum buffer type, pH, buffer concentration, working potential, temperature and amounts of enzyme and mediator for PVF modified enzyme electrode were found to be phosphate, 8.0, 0.05 M, +0.70 and +0.30 V, 40 °C, 2.0 Unit and 10.9%, respectively. The repeatability, storage stability of the enzyme electrodes and interference effects were also investigated. Enzyme electrodes were used for determination of uric acid in serum samples and the results were in a good agreement with those obtained by commercial enzymatic kits.

Integrated multienzyme electrochemical biosensors for monitoring malolactic fermentation in wines

Integrated amperometric biosensors for the determination of L-malic and L-lactic acids were developed by coimmobilization of the enzymes L-malate dehydrogenase (MDH) and diaphorase (DP), or L-lactate oxidase (LOX) and horseradish peroxidase (HRP), respectively, together with the redox mediator tetrathiafulvalene (TTF), on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +100mV (vs. Ag/AgCl), and the reduction of TTF(+) at -50mV were used for the monitoring of the enzyme reactions involved in L-malic and L-lactic acid determinations, respectively. Experimental variables concerning the biosensors composition and the detection conditions were optimized for each biosensor. Good relative standard deviation values were obtained in both cases for the measurements carried out with the same biosensor, with no need of cleaning or pretreatment of the bioelectrodes surface, and with different biosensors constructed in the same manner. After 7 days of continuous use, the MDH/DP biosensor still exhibited 90% of the original sensitivity, while the LOX/HRP biosensor yielded a 91% of the original response after 5 days. Calibration graphs for L-malic and L-lactic were obtained with linear ranges of 5.2x10(-7) to 2.0x10(-5) and 4.2x10(-7) to 2.0x10(-5)M, respectively. The calculated detection limits were 5.2x10(-7) and 4.2x10(-7)M, respectively. The biosensors exhibited a high selectivity with no significant interferences. They were applied to monitor malolactic fermentation (MLF) induced by inoculation of Lactobacillus plantarum CECT 748(T) into a synthetic wine. Samples collected during MLF were assayed for L-malic and L-lactic acids, and the results obtained with the biosensors exhibited a very good correlation when plotted against those obtained by using commercial enzymatic kits.

Enzymatic activity of lipase in post-metamorphic phase bullfrogs

The knowledge of the digestive system of bullfrogs is an important step for the determination of their nutritional requirements throughout growth phases. With the objective of evaluating the enzymatic activity of lipase in the intestinal content of bullfrogs (Rana catesbeiana Shaw, 1802), 100 animals with median weight of 3.6 g were distributed in stalls under controlled temperature and photoperiod. The frogs, selected at the post-metamorphic phase, received commercial extruded diet ad libitum throughout the 87-day experiment. The collections of the intestinal content were performed by the desensitization of the frogs in ice and water at 0ºC and subsequent isolation of the small intestine. Determination of lipase activity was performed with a commercial enzymatic kit (Lipase-Bioclin, MG, Brazil), first measured in samples taken at day three (3.46 UI). During the initial phase the bullfrog possesses low lipase hydrolysis capacity was found, having a specific activity of 217 UI mg-1. In the subsequent period both lipase activity and specific lipase activity continuously increased. Lipase activity as a function of bullfrog weight fell after day twenty and reached 0.33 UI g-1, for frogs of medium weight (179 g). Feed for bullfrogs at the post-metamorphic phase weighing more than 10 g can have larger amounts of ingredients containg lipids, due to the increased digestive capacity of these frogs.

Atividade da amilase em rã-touro durante a fase pós-metamórfica

O objetivo deste trabalho foi avaliar a atividade da amilase no quimo da rã-touro, Rana catesbeiana Shaw 1802 (3,6 a 200 g). Oitenta e sete animais foram distribuídos em baias-testes com temperatura e fotoperíodo controlados. As rãs selecionadas na fase pós-metamórfica receberam ração comercial extrusada ad libitum. Durante 87 dias de experimento, foram efetuadas 29 coletas em intervalos de 1 a 8 dias. As coletas do conteúdo intestinal foram feitas mediante a insensibilização das rãs em gelo e água e isolamento posterior do intestino delgado das mesmas. Para os testes da atividade da amilase, foram utilizados kits enzimáticos comerciais. A atividade da amilase foi observada a partir do terceiro dia e apresentou aumento até alcançar estabilidade, quando os animais pesavam 4,35 g. Do sétimo dia até o final do experimento, ocorreu a manutenção da estabilidade da atividade (137,44 UI) e atividade específica da amilase (14.688 UI mg-1).

A procedure for measuring triacylglyceride and cholesterol content using a small amount of tissue

This paper describes the development of a procedure for measuring total lipid, triacylglyceride, and cholesterol content using small amounts of tissue (50 mg for liver samples and 10 mg for adipose tissue samples). In the first step, total lipids are extracted with an organic solvent mixture of hexane and isopropanol. In the second step, extracted lipids are emulsified by sonication in a buffer containing 1,4-piperazinediethanesulfonic acid (28.75 mM), magnesium chloride·6H 2O (57.76 mM), free fatty acids–bovine serum albumin (8.76 μM), and sodium dodecyl sulfate (0.1%). Lipid concentrations from the resulting emulsion can then be assessed using commercial enzymatic kits. This method is also suitable for determining direct homogenate triacylglyceride and total cholesterol content in cases where a lipid percentage is not needed. The proposed method increases the yield of lipid recovery from small tissue samples and allows the measurement of both triacylglyceride and cholesterol content from a single starting sample. The methodology described here is the first to accomplish simultaneous determination of both parameters and is potentially useful for animal small tissue samples, particularly human biopsy samples.

Comparative study of two commercial enzymatic kits for determining oxalate concentrations in urine

We performed a comparative study of two commercial kits for determining oxalate in urine. These were: (a) an oxalate decarboxylase-based assay (Boehringer Mannheim); (b) an oxalate oxidase-based assay (Sigma). The within-run and between-run imprecision were found to be similar in both methods. The recovery was 94% with the oxalate decarboxylase method. The pH of the specimen had a major effect on the recovery obtained by the oxalate oxidase method (66–86% at pH = 2.5 and 37–68% at pH = 1.5. The decarboxylase method was linear up to at least 2224 μmol/L and the oxidase method was linear up to at least 890 μmol/L. We also studied the interference of ascorbic acid in both techniques and found a positive bias with the oxidase method and a negative bias using the decarboxylase method. The correlation coefficient was 0.592.

Microplate measurement of urinary albumin and creatinine.

We describe microplate methods for measurement of human urinary albumin (HUA) by competitive enzyme-linked immunosorbant assay (ELISA) and creatinine with a modified commercial enzymatic kit. Incorporation of substrate mixing into the competitive ELISA changes the dynamic absorbance-concentration response, greatly simplifying calculations and improving sensitivity and accuracy. Measurement of creatinine in urine and plasma samples with a commercially available enzymatic kit modified for analysis by use of an inexpensive microplate reader produced values comparable in precision and accuracy to those obtained by an automated kinetic Jaffé method.

Screening for cerebrotendinous xanthomatosis by using an enzymatic assay for 7 alpha-hydroxylated steroids in urine.

We used a commercial enzymatic kit for measuring 7 alpha-hydroxylated bile acids to screen urines from normal subjects, liver-transplant recipients, and patients with various liver diseases, cerebro-hepato-renal syndrome, or cerebrotendinous xanthomatosis (CTX). Because of their high concentrations of 7 alpha-hydroxylated compounds excreted, the CTX patients were clearly distinguished from all other groups except for a slight overlap with the patients with cerebro-hepato-renal syndrome and liver-transplant recipients. Gas chromatography for bile alcohols completed the differential diagnosis.

Quantitation method for choline-containing phospholipids in human serum lipoproteins by high performance liquid chromatography.

A determination method for choline-containing phospholipids in serum lipoproteins was established by application of the HPLC method [Hara et al. (1980) J. Biochem. 87, 1863-1865; (1981) 89, 879-887] developed for cholesterol quantitation. The concentration of choline-containing phospholipids in the fraction separated by HPLC using gel permeation columns was determined through colorimetric detection using a commercial enzymatic kit. The optimum conditions for enzymatic reaction in the flow diagram were examined. The concentration calculated from the A500 peak area was found to reflect very precisely the concentration of choline-containing phospholipids in all lipoprotein fractions, and the quantitation of each lipoprotein fraction can be performed with only 10-20 microliters of whole serum. Monitoring the elution patterns by detecting choline-containing phospholipids can give much more information about lipoprotein distributions according to particle size than analyses done by detecting cholesterol.