A rapid method for the determination of CTP and phosphocholine in rat liver and baby hamster kidney 21 cells

1978 ◽  
Vol 56 (8) ◽  
pp. 831-835 ◽  
Author(s):  
Patrick C. Choy ◽  
Frederick W. Whitehead ◽  
Dennis E. Vance
Keyword(s):  
Rat Liver ◽  
Inorganic Pyrophosphatase ◽  
Baby Hamster Kidney ◽  
Sensitive Assay ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Phosphatidylcholine Biosynthesis ◽  
Ammonium Sulfate Precipitate ◽  
Cytosolic Extract ◽  
Wet Weight

A rapid and sensitive assay for CTP and phosphocholine was required for us to determine the concentration of these compounds in tissues and cell cultures. Such a procedure was devised with CTP:phosphocholine cytidylyltransferase, an enzyme which is highly specific for CTP and phosphocholine. The 0–22% ammonium sulfate precipitate of a cytosolic extract from rat liver was used as the source of the enzyme. The amount of CTP in an extract was estimated by the conversion of [3H]phosphochoiine to 3H-labelled CDP-choline. Similarly, the concentration of phosphocholine was estimated by the formation of 3H-labelled CDP-choline from 3H-labelled CTP. The conversion of CTP and phosphocholine to CDP-choline was 90% when inorganic pyrophosphatase was added to the incubations. The formation of CDP-choline was linear between 1 and 10 nmol of CTP or phosphocholine. The concentration of CTP was determined in rat liver (62 nmol/g wet weight) and baby hamster kidney 21 (BHK-21) cells (161 nmol/g wet weight). The concentration of phosphocholine in rat liver was 1.16 μmol/g wet weight whereas in BHK-21 cells it was much less (69 nmol/g wet weight). By this procedure, it may be possible to establish the importance of CTP and phosphocholine in the control of phosphatidylcholine biosynthesis.

scholarly journals Effect of hypoxia on phosphatidylcholine biosynthesis in the isolated hamster heart

1990 ◽  
Vol 268 (1) ◽  
pp. 47-54 ◽  
Author(s):  
G M Hatch ◽  
P C Choy
Keyword(s):  
Active Form ◽  
Compensatory Mechanism ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Hypoxic Conditions ◽  
Rate Limiting Step ◽  
Phosphatidylcholine Biosynthesis ◽  
Hamster Heart ◽  
Rate Limiting ◽  
Hypoxic Treatment

In hamster heart, the majority of the phosphatidylcholine is synthesized via the CDP-choline pathway, and the rate-limiting step of this pathway is catalysed by CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15). We have shown previously [Choy (1982) J. Biol. Chem. 257, 10928-10933] that, in the myopathic heart, the level of cardiac CTP was diminished during the development of the disease. In order to maintain the level of CDP-choline, and consequently the rate of phosphatidylcholine biosynthesis, cardiac cytidylyltransferase activity was increased. However, it was not clear if the same compensatory mechanism would occur when the cardiac CTP level was decreased rapidly. In this study, hypoxia of the hamster heart was produced by perfusion with buffer saturated with 95% N2. The heart was pulse-labelled with radioactive choline and then chased with non-radioactive choline for various periods under hypoxic conditions. There was a severe decrease in ATP and CTP levels within 60 min of hypoxic perfusion, with a corresponding fall in the rate of phosphatidylcholine biosynthesis. Analysis of the choline-containing metabolites revealed that the lowered ATP level did not affect the phosphorylation of choline to phosphocholine, but the lower CTP level resulted in the decreased conversion of phosphocholine to CDP-choline. Determination of enzyme activities revealed that hypoxic treatment resulted in the enhanced translocation of cytidylyltransferase from the cytosolic to the microsomal form. This enhanced translocation was probably caused by the accumulation of fatty acids in the heart during hypoxia. We postulate that the enhancement of translocation of the cytidylyltransferase to the microsomal form (a more active form) is a mechanism by which the heart can compensate for the decrease in CTP level during hypoxia in order to maintain phosphatidylcholine biosynthesis.

Activities of the phosphatidylcholine biosynthetic enzymes in rat liver during development

1983 ◽  
Vol 61 (10) ◽  
pp. 1147-1152 ◽  
Author(s):  
Steven L. Pelech ◽  
Ellen Power ◽  
Dennis E. Vance
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Specific Activity ◽  
Perinatal Period ◽  
Choline Kinase ◽  
Specific Enzyme ◽  
Methyltransferase Activity ◽  
Specific Enzyme Activity ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Phosphatidylcholine Biosynthesis

The activities of the enzymes of rat hepatic phosphatidylcholine biosynthesis have been measured as a function of development in the rat (term, 23 days). During the last 5 days of gestation, the specific activity of choline kinase was elevated almost fivefold (p < 0.05). After parturition, choline kinase activity was reduced to adult values by the 5th postnatal day. Over 75% of the total CTP:phosphocholine cytidylyltransferase protein in prenatal liver was detected in the cytosolic fraction. On the day of birth, most of the cytidylyltransferase translocated to the microsomes so that the microsomal specific enzyme activity was 3.3-fold higher (p < 0.01) and the cytosolic specific enzyme activity (measured in the presence of phospholipid) was 68% lower (p < 0.001) than the day before parturition. CDPcholine:diacylglycerol cholinephosphotransferase activity (measured in the presence of diacylglycerol) increased 130-fold (p < 0.001) during the last 5 days of gestation. On the 10th postnatal day, cholinephosphotransferase activity was 1.7-fold higher (p < 0.001) than immediately after birth, but declined to adult values by the 19th day. Between the 5th day prior to parturition and the 10th postnatal day, phosphatidylethanolamine N-methyltransferase activity steadily increased 16-fold (p < 0.001). The results are in agreement with the hypothesis that the increase in phosphatidylcholine in rat liver during the perinatal period is due to an increased synthesis of CDPcholine, which is a consequence of the translocation of the cytidylyltransferase from cytosol to the endoplasmic reticulum.

BIOASSAY OF PROLACTIN

1969 ◽  
Vol 60 (1) ◽  
pp. 91-100 ◽  
Author(s):  
Charles S. Nicoll
Keyword(s):  
Epithelial Cells ◽  
Lipid Content ◽  
Dry Weight ◽  
Fat Content ◽  
Assay Sensitivity ◽  
Mucosal Epithelium ◽  
Wet Weight ◽  
Total Dry Weight

ABSTRACT The response of the pigeon crop-sac to systemically acting prolactin (injected subcutaneously) was evaluated by measuring the wet weight of the responsive lateral lobes of the organ and by determining the dry weight of a 4 cm diameter disc of mucosal epithelium taken from one hemicrop. Of several different injection schedules tested, administration of prolactin in four daily injections was found to yield optimal responses. When compared with a graded series of prolactin doses, measurement of the mucosal dry weight proved to be a better method of response quantification than determination of the crop-sac wet weight with respect to both assay sensitivity and precision. The submucosal tissue of the crop-sac was estimated to constitute about 64 % of the total dry weight of the unstimulated organ and it was found to be relatively unresponsive to prolactin stimulation in comparison with the mucosa. The lipid content of the mucosal epithelium was determined using unstimulated crop-sacs or tissues which showed varying degrees of prolactin-induced proliferation. The fat content of the mucosal epithelial cells increased only slightly more rapidly than the dry weight or the defatted dry weight of the mucosa. Suggestions are made for the further improvement of the systemic crop-sac assay for prolactin.

scholarly journals Experimental model for in vivo determination of dietary fibre and its effect on the absorption of nutrients in the small intestine

1981 ◽  
Vol 45 (2) ◽  
pp. 283-294 ◽  
Author(s):  
Ann-Sofie Sandberg ◽  
H. Andersson ◽  
B. Hallgren ◽  
Kristina Hasselblad ◽  
B. Isaksson ◽  
...  
Keyword(s):  
Small Intestine ◽  
Wheat Bran ◽  
Dietary Fibre ◽  
Dry Weight ◽  
Direct Analysis ◽  
Fresh Weight ◽  
Wet Weight ◽  
Definition Of

1. An experimental model for the determination of dietary fibre according to the definition of Trowell et al. (1976) is described. Food was subjected to in vivo digestion in ileostomy patients, and the ileostomy contents were collected quantitatively, the polysaccharide components of which were analysed by gas–liquid chromatography and the Klason lignin by gravimetric determination. The model was used for the determination of dietary fibre in AACC (American Association of Cereal Chemists), wheat bran and for studies on the extent of hydrolysis of wheat-bran fibre in the stomach and small intestine. The effect of wheat bran on ileostomy losses of nitrogen, starch and electrolytes was also investigated.2. Nine patients with established ileostomies were studied during two periods while on a constant low-fibre diet. In the second period 16 g AACC wheat bran/d was added to the diet. The ileostomy contents and duplicate portions of the diet were subjected to determinations of wet weight, dry weight, water content, fibre components, starch, N, sodium and potassium.3. The wet weight of ileostomy contents increased by 94 g/24 h and dry weight by 10 g/24 h after consumption of bran. The dietary fibre of AACC bran, determined as the increase in polysaccharides and lignin of ileostomy contents after consumption of bran, was 280 g/kg fresh weight (310 g/kg dry matter). Direct analysis of polysaccharides and lignin in bran gave a value of 306 g/kg fresh weight. Of the added bran hemicellulose and cellulose 80–100% and 75–100% respectively were recovered in ileostomy contents. There was no significant difference between the two periods in amount of N, starch and K found in the ileostomy contents. The Na excretion increased during the ‘bran’ period and correlated well with the wet weight of ileostomy contents.4. In conclusion, it seems probable that determination of dietary fibre by in vivo digestion in ileostomy patients comes very close to the theoretical definition of dietary fibre, as the influence of bacteria in the ileum seems small. Bacterial growth should be avoided by using a technique involving the change of ileostomy bags every 2 h and immediate deep-freezing of the ileostomy contents. True dietary fibre can be determined by direct analysis of polysaccharides and lignin in the food, at least in bran. Very little digestion of hemicellulose and cellulose from bran occurs in the stomach and small bowel. The 10–20% loss in some patients may be due to digestion by the gastric juice or to bacterial fermentation in the ileum, or both. The extra amount of faecal N after consumption of bran, reported by others, is probably produced in the large bowel.

scholarly journals An improvement of calcium determination technique in the shell of molluscs

2009 ◽  
Vol 52 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Cristiane Soído ◽  
Maurício Carvalho Vasconcellos ◽  
Antônia Gonçalves Diniz ◽  
Jairo Pinheiro
Keyword(s):  
Hydrogen Peroxide ◽  
Nitric Acid ◽  
Experimental Model ◽  
Calcium Content ◽  
Conventional Technique ◽  
Organic Substances ◽  
Complexometric Method ◽  
Wet Weight ◽  
Calcium Determination

The complexometric method is usually applied to quantitative calcium determination in different materials; however the application of this method to calcium determination in molluscs shells infers significant interferences to the results. The snail Bradybaena similaris, a terrestrial gastropod, was used as experimental model to the improvement of this method. The shells were calcinated and dissolved in nitric acid, the hydrogen peroxide was also used to clarify the medium after the acid addition. The calcination procedure and the use of nitric acid reduced the significantly the interferences, allowing a major degree of destruction of the organic substances of the shell. The improvement of the calcium determination technique usually employed showed calcium content of 874.24 ± 56.617 mg of CaCO3/g of ash in comparison to the conventional technique that allowed the determination of 607.79 ± 67.751 mg of CaCO3/g of shell, wet weight.

scholarly journals CYTOFLUOROMETRIC DETERMINATION OF LYSINE WITH DANSYLCHLORIDE

1968 ◽  
Vol 16 (7) ◽  
pp. 459-466 ◽  
Author(s):  
A. ROSSELET ◽  
F. RUCH
Keyword(s):  
Rat Liver ◽  
Lysine Content ◽  
Amino Groups ◽  
Isolated Rat Liver ◽  
Model Experiments ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Isolated Rat

Dansylchloride (l-dimethylamino-naphthalene-5-sulfochloride) may be used for cytofluorometric determination of lysine. By means of model experiments on protein smears it is shown that the reaction must be carried out in ethanol if it is to be specific for amino groups. The fluorescence given by isolated rat liver nuclei treated with dansylchloride corresponds to the three classes of 2n, 4n and 8n nuclei. The dansylfluorescence of several kinds of sperms is proportional to their lysine content. In rat liver nuclei, 95% of the lysine is dansylated and the lysine content may be determined in absolute values by comparison with polylysine. In spermatozoa only 50% of the lysine reacts.

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