Method for triglyceride measurement in small amounts of plasma

A method is described for determining triacylglyceride concentration in small amounts of plasma. After ethanolic-KOH digestion of diluted plasma aliquots, samples were neutralized with MgSO4 and glycerol was fluorimetrically assayed in supernatants by the coupling of glycerokinase, pyruvate kinase, and lactate dehydrogenase catalyzed reactions. Values were corrected by free glycerol present in the non-digested samples. Digestions were performed at different times and temperatures in order to establish optimum conditions for recoveries and reproducibility. Parallel determinations before and after phospholipid removal showed that their presence in plasma did not interfere with the obtained values. This method is especially useful for running many samples in parallel and for determinations in small experimental animals in which the amount of plasma is very limited.     

Heritability of plasma cholesterol and triglyceride concentrations in swine

Three-hundred and eighteen female swine representing contemporary commercial swine breeds (34 Chester White, 43 Large White, 42 Landrace, 40 Yorkshire, and 159 four-breed crossbreeds) were used to evaluate genetic variation between and within breeds for levels of plasma cholesterol and plasma triglycerides. Blood was sampled from all pigs after a 16-hr fast at 154 days of age. Plasma cholesterol was measured in all pigs and triglycerides were measured in 232 pigs. Paternal half-sib heritabilities (h2) for plasma cholesterol and plasma triglycerides were 0.45 +/- 0.23 and 1.04 +/- 0.32, respectively. Breed differences were not apparent for either trait. Phenotypic and paternal half-sib genetic correlations between the two traits were 0.16 and 0.66, respectively. Neither body weight nor backfat depth were important in affecting the estimate of h2 for either trait. The relatively high h2 of total plasma cholesterol and of total triglycerides offers the possibility of developing, through selection, populations of hypercholesterolemic or hypertriglyceridemic swine useful as models for studies directed toward further understanding of human cardiovascular disease.     

Fluorometric determination of plasma adenosine concentrations using high-performance liquid chromatography

Methods are described for the fluorometric determination of plasma adenosine concentrations, using HPLC. Plasma obtained from blood of dogs treated with erythro-(2-hydroxy-3-nonyl)adenine hydrochloride and dipyridamole was deproteinized with perchloric acid and the neutralized sample was put sequentially onto a SepPak C18 and boronic acid affinity column. Subsequently, adenosine in the final elution was converted to 1,N6-ethenoadenosine and was quantitated by HPLC with a fluorescence detector. The percentage recovery of adenosine added to the deproteinized plasma was nearly 100%. In the adenosine deaminase treated plasma, the increase in adenosine concentration of even 4 nM can be accurately determined. The control renal venous plasma concentrations of adenosine in anesthetized dogs were 19.9 +/- 1.9 nM, a significantly higher value than the corresponding arterial concentrations (12.7 +/- 1.1 nM), thereby suggesting the renal release of adenosine. This release was markedly enhanced following the removal of the renal arterial occlusion. Thus, taken together with the in vivo results, the present method is sensitive, hence most useful for the determination of plasma adenosine concentrations.     

Extraction and labeling methods for microarrays using small amounts of plant tissue

Procedures were developed to maximize the yield of high-quality RNA from small amounts of plant biomass for microarrays. Two disruption techniques (bead milling and pestle and mortar) were compared for the yield and the quality of RNA extracted from 1-week-old Arabidopsis thaliana seedlings (approximately 0.5–30 mg total biomass). The pestle and mortar method of extraction showed enhanced RNA quality at the smaller biomass samples compared with the bead milling technique, although the quality in the bead milling could be improved with additional cooling steps. The RNA extracted from the pestle and mortar technique was further tested to determine if the small quantity of RNA (500 ng–7 μg) was appropriate for microarray analyses. A new method of low-quantity RNA labeling for microarrays (NuGEN Technologies, Inc.) was used on five 7-day-old seedlings (approximately 2.5 mg fresh weight total) of Arabidopsis that were grown in the dark and exposed to 1 h of red light or continued dark. Microarray analyses were performed on a small plant sample (five seedlings; approximately 2.5 mg) using these methods and compared with extractions performed with larger biomass samples (approximately 500 roots). Many well-known light-regulated genes between the small plant samples and the larger biomass samples overlapped in expression changes, and the relative expression levels of selected genes were confirmed with quantitative real-time polymerase chain reaction, suggesting that these methods can be used for plant experiments where the biomass is extremely limited (i.e. spaceflight studies).     

Quantitative measurement of starch in very small amounts of leaf tissue

Summary A specific enzyme method is described for the routine estimation of starch in small quantities (10–30 mg) of dried leaf tissue. A -glucanase-free preparation of amyloglucosidase is employed to hydrolyse starch to glucose; this is subsequently estimated by the glucose oxidase technique. The method gives result which agree closely with those obtained by a specific iodine-precipitation method.     

Mitochondrial DNA and RNA isolation from small amounts of potato tissue

We present a fast and simple protocol for purification of mitochondrial DNA and RNA from small amounts of potato tissue including tubers, leaves, flowers, and flower buds. This method uses a high ionic strength medium to isolate mitochondria and extract mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. The mitochondrial DNA was not contaminated by plastid DNA, was fully restrictable and was successfully used for PCR, cloning and Southern analyses. Similarly, the isolated mitochondrial RNA was not contaminated (flower buds) or only slightly contaminated (leaves) by plastid RNA. RNA prepared according to our method was acceptable for northern and RT-PCR analyses.     

Subtractive hybridization of cDNA from small amounts of plant tissue

A subtractive hybridization method is described that allows the generation of a subtractive gene library from small amounts of plant or other eukaryotic tissues. The method uses paramagnetic oligo-dT beads to capture poly-adenylated mRNA and to synthesize the complementary cDNA on a solid support. The use of magnetic beads facilitates the change of reaction buffers and the removal of primers and minimizes yield losses. Subtracted material obtained from this method can either be cloned directly or used to screen a specific library.     

Normative standards of plasma cholesterol and triglyceride concentrations in Canadians of working age.

Fasting plasma cholesterol and triglyceride concentrations were determined for 6407 working Canadian adults aged 20 to 69 years in Toronto and Hamilton. Means, medians and 5th and 95th percentiles were ascertained from the data for men, women taking oral contraceptives or estrogen preparations, and women not taking such medication. Mean plasma cholesterol values (mg/dL) ranged in men from 168.3 at ages 20 to 24 years to 211.5 at ages 45 to 49 years, and in women using hormone preparations from 180.3 at ages 20 to 24 years to 224.2 at ages 50 to 54 years; corresponding values in women not using these preparations were 164.9 and 220.6. Plasma triglyceride means (mg/dL) ranged in men from 108.7 at ages 20 to 24 years to 166.7 at ages 40 to 44 years, in women using hormone preparations from 115.4 at ages 20 to 24 years to 145.3 at ages 45 to 59 years, and in women not using these preparations from 77.5 at ages 20 to 24 years to 112.4 at ages 50 to 54 years.     

A simple rapid method for ganglioside isolation from small amounts of tissue.

Gangliosides from as little as 1 mg dry wt of brain tissue can be isolated for thin-layer chromatography by a simple, rapid method which combines extraction by chloroformmethanol with a single step silicic acid column separation of gangliosides from the bulk of nonganglioside lipids.     

Fluorometric assay for tissue transglutaminase-mediated transamidation activity

Herein, we report the development of a direct discontinuous fluorometric transamidation assay for determining tissue transglutaminase (TG2) activity. In the assay reaction, TG2 catalyzes the formation of a biotin-fluorophore conjugate, using a fluorescent, high affinity γ-glutamyl donor substrate and a biotinylated amine as a γ-glutamyl acceptor substrate. After the reaction, the conjugate is fixed on streptavidin-coated beads and excess substrate is washed away, allowing the transamidation activity to be quantified by fluorescence measurement. This method was used to detect the activity of as little as 0.6 mU of purified TG2, and can be used for detection of activity from crude cellular lysates. Furthermore, this assay can be used for screening potential inhibitors and synthetic substrates, the latter of which was demonstrated herein.