Glycosaminoglycan content in tissues and body fluids of rats treated with atherogenic diet.

An increase of total glycosaminoglycan content in aortic wall and liver as well as changes in the concentration of glycosaminoglycan fractions in aorta, skin, liver, and blood serum were found in white rats fed with atherogenic diet. Urinary excretion of glycosaminoglycans was increased in experimental animals.     

Phosphorylase activity and glycogen content in cerebral and myocardium tissues in rats of different age

The phosphorylase activity (EC 2.4.1.1) and the glycogen content were determined in the brain and myocardium of adult and old rats. In the cortex and while substance of great cerebral hemispheres, the phosphorylase a activity decreases and the phosphorylase b activity increases, while the total enzyme activity remains unchanged with aging. In the myocardium, the activity of phosphorylase a increases, while that of phophorylase b decreases against a background of an insignificant decrease in the total phosphorylase activity. During aging, the glycogen content decreases sharply in the myocardium, whereas in the cerebral tissue it remains unchanged. Age peculiarities of the adrenaline effect upon myocardial phosphorylase activity are presented.     

Radioimmunoassay for biopterin in body fluids and tissues

Specific antibodies against l-erythro-biopterin have been prepared in rabbits using the conjugates to bovine serum albumin. The antiserum against l-erythro-biopterin distinguished among l-erythro-tetrahydro- or 7,8-dihydro-biopterin, the other three stereoisomers of biopterin, d-erythro-neopterin, folic acid, and other synthetic pteridines. Using the specific antiserum against l-erythro-biopterin, a radioimmunoassay has been developed to measure the biopterin concentrations in urine, serum, cerebrospinal fluid, and tissues. The conjugate of l-erythro-biopterin with tyramine, 4-hydroxy-2-[2-(4-hydroxyphenyl)ethylamino]-6-(l-erythro-1,2-dihydroxypropyl)pteridine (BP-TYRA), was synthesized and labeled with ¹²⁵I as the labeled ligand for the radioimmunoassay. BP-¹²⁵I-TYRA had similar binding affinity as the natural l-erythro-biopterin and was thus permitted to establish a highly sensitive radioimmunoassay for biopterin. The limit of sensitivity of the radioimmunoassay with BP-¹²⁵I-TYRA as labeled ligand was 0.5 pmol. The total concentration of biopterins, i.e., biopterin, 7,8-dihydro-, quinonoid dihydro and tetrahydrobiopterins, in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, whereas iodine oxidation under alkaline conditions gave the concentration only of the former two. Biopterin in urine could be measured directly using 1 μl of urine, but a pretreatment with a small Dowex 50-H⁺ column was required for serum, cerebrospinal fluid, and brain tissues.     

Radioimmunoassay for neopterin in body fluids and tissues

Specific antibodies against D-erythroneopterin have been prepared in rabbits using a conjugate of D-erythroneopterin to bovine serum albumin (D-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished D-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, L-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for D-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of D-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic nucleotide activity in gastrointestinal tissues and fluids.

Published values of adenosine 3′,5′-monophosphate (cAMP) and guanosine 3′,5′-monophosphate (cGMP) in gastrointestinal tissues and fluids have varied depending upon extraction and purification methods and assay procedures. Cyclic nucleotide levels in a variety of gastrointestinal tissues and fluids from several animal species were measured by the Gilman protein kinase assay for cAMP, and by a radioimmunoassay for cAMP and cGMP. The neutral alumina/Dowex-1-formate column was the most accurate for the preparation of bile and gastric juice samples for the measurement of cAMP and cGMP, as verifled by the use of column blanks, spiked samples, phosphodiesterase treatment, and serial sample dilution. Tissue samples gave consistent results regardless of the various column procedures employed.     

Recent advances in protein profiling of tissues and tissue fluids

Creating protein profiles of tissues and tissue fluids, which contain secreted proteins and peptides released from various cells, is critical for biomarker discovery as well as drug and vaccine target selection. It is extremely difficult to obtain pure samples from tissues or tissue fluids, however, and identification of complex protein mixtures is still a challenge for mass spectrometry analysis. Here, we summarize recent advances in techniques for extracting proteins from tissues for mass spectrometry profiling and imaging. We also introduce a novel technique using a capillary ultrafiltration (CUF) probe to enable in vivo collection of proteins from the tissue microenvironment. The CUF probe technique is compared with existing sampling techniques, including perfusion, saline wash, fine-needle aspiration and microdialysis. In this review, we also highlight quantitative mass spectrometric proteomic approaches with, and without, stable-isotope labels. Advances in quantitative proteomics will significantly improve protein profiling of tissue and tissue fluid samples collected by CUF probes.     

Measurement of inorganic pyrophosphate in biological fluids and bone tissues

A simple and rapid technique of measuring pyrophosphates in plasma, urine, and bone tissue is described, using the UDPG-pyrophosphorylase reaction and fluorimetrical determination of NADPH formed in a combined system of phosphorylation and reduction. This very specific method avoids the separation of Pi² by column chromatography, and its very great sensitivity enables measurement on a final sample corresponding to 0.2 ml of plasma, with a precision of about 3%.The mean plasma PPi concentration (±SE of mean) was 3.53 ± 0.19 μmoles/liter (SE=0.93), or 0.207 ± 0.006 mg Pi/liter. The normal range for this population (99% confidence limit) is therefore between 1.10 and 5.90 μmoles/liter or 0.068 – 0.366 mg Pi/liter. Analysis of the variation of the duplicate measurements shows a very small divergence of 3.4% or ±0.12 μm.Normal 24 hr urinary excretion is 53.0 ± 6.8 μmoles with 99% confidence limits of 10.0 and 96.0 μmoles.The average PPi concentration in calvaria of 20 10-day-old rats is 3.685 ± 0.16 nmoles/mg fresh weight.     

Identification and characterization of melanin in tissues and body fluids.

A new method for the unambiguous identification of melanin in biological materials has been developed. It may also be used to differentiate between melanins from various tissues and with various properties. The method is based on the detection and characterization of the free radicals in melanin by Electron Spin Resonance Spectroscopy. Applications of this approach include the identification of microscopically undetectable melanin in amelanotic melanomas and identification of the nature of the pigment in the Dubin-Johnson Syndrome.     

A GC-MS/MS method for the quantitative analysis of low levels of the tyrosine metabolites maleylacetone, succinylacetone, and the tyrosine metabolism inhibitor dichloroacetate in biological fluids and tissues

We developed a sensitive method to quantitate the tyrosine metabolites maleylacetone (MA) and succinylacetone (SA) and the tyrosine metabolism inhibitor dichloroacetate (DCA) in biological specimens. Accumulation of these metabolites may be responsible for the toxicity observed when exposed to DCA. Detection limits of previous methods are 200 ng/mL (1.2 pmol/microL) (MA) and 2.6 microg/mL (16.5 pmol/microL) (SA) but the metabolites are likely present in lower levels in biological specimens. To increase sensitivity, analytes were extracted from liver, urine, plasma and cultured nerve cells before and after dosing with DCA, derivatized to their pentafluorobenzyl esters, and analyzed via GC-MS/MS.     

Acetoin metabolism in animal tissues

The acetoin-synthesizing activity has been studied in the skeletal muscles, brain, liver and spleen homogenates (numbered as the activity decreases). The acetoin-synthesizing activity drastically increases in case of the acetaldehyde excess and alcohol intoxication. The acetaldehyde concentrations of above 1.10(-3) M inhibit the liver pyruvate dehydrogenase activity and increase the non-oxidative transformation of pyruvate. Acetoin is rapidly metabolized in the organism eliminating from blood 10 minutes after its injection. Acetoin is an effective precursor in the biosynthesis of lipids.     

Sialic acid content of low density lipoprotein and its relation to lipid concentrations and metabolism of low density lipoprotein and cholesterol

A low sialic acid content in low density lipoprotein (LDL) has been associated with atherogenicity and coronary artery disease (CAD) in many but not all studies. We investigated associations of the sialic acid-to-apolipoprotein B (apoB) ratio of LDL with lipoprotein lipid concentrations, kinetics of LDL, metabolism of cholesterol, and the presence of CAD in 98 subjects (CAD(+), n = 56; CAD(-), n = 42). The sialic acid ratios of total, dense, and very dense LDL were lower in the CAD(+) than CAD(-) subjects, especially at high sialic acid ratios. The LDL sialic acid ratio was inversely associated with respective lipid and apoB concentrations and positively with lipid-to-apoB ratios of LDL. The transport rates (TRs) for total and dense LDL apoB were negatively associated with their sialic acid ratios. The sialic acid ratio of dense LDL, but not that of total LDL, was inversely correlated with serum levels of cholesterol precursor sterols, indicators of cholesterol synthesis, and positively with serum levels of plant sterols, indicators of cholesterol absorption. In addition, the TR for dense LDL was positively correlated with cholesterol synthesis.In conclusion, a low LDL sialic acid ratio was associated with CAD, high numbers of small LDL particles, and a high TR for LDL apoB, and in dense LDL also with high synthesis and low absorption of cholesterol.