Phosphorus Assay Research Articles

Calcium-Binding Proteins in Serum of Chickens: Vitellogenin and Albumin

Two Ca-binding proteins of hen serum were resolved by gel filtration in45Ca buffer and were named CaBP(l) and CaBP(2). The major calcium-binding protein, CaBP(l), had a molecular size of 6.0 X 105 daltons and appears to be vitellogenin. Deeley et al. (1975) described vitellogenin as the precursor of lipovitellin and phosvitin, the Ca-binding proteins of egg yolk. The present paper demonstrates that “native” vitellogenin is a calcium-binding protein in the serum of laying chickens. The CaBP(2) co-eluted with and was immunologically identical to chicken serum albumin which also bound 45Ca. Data from elution profiles and phosphorus assays indicated that CaBP(l) and CaBP(2) were different from phosvitin and the Ca-binding proteins of the duodenum and uterus.

Interference of polyols with the Lowry-Lopez phosphorus assay: Reaction of polyols with Mo 7O 246−

Certain open-chain polyols were shown to interfere with the determination of phosphorus of the Lowry-Lopez method by forming a complex with Mo 7O 24 6−. The ability to interfere with the assay increased with increasing chain length of the polyols: Ethylene glycol and glycerol did not react at all; i-erythritol reacted to a small extent, but hexitols and perseitol formed stronger complexes. Depending on the polyol, interference occurred even at 0.2 m m (hexitols) or 2 m m (xylitol) concentrations. At these concentrations the polyols interfered only to a small extent with the phosphorus assays based on the use of Triton X-100 and molybdate. The complex formation was exploited in the development of a colorimetric polyol assay.

Digestion and absorption of lecithin in larvae of the cabbage butterfly, Pieris brassicae

1. 1. Dietary lecithin [phosphatidyl ( N-methyl- 14C) choline] is converted to the surface-active lysolecithin and to water-soluble metabolites in the intestinal lumen of the butterfly, Pieris brassicae. 2. 2. In the midgut cells radioactivity was found in lecithin, lysolecithin and sphingomyelin. 3. 3. In the haemolymph over 90% of the radioactivity was water-soluble after 6hr, but only 32% was water-soluble after 48 hr of feeding. Labeled lipids in the haemolymph were lecithin ( ca. 90% of lipid radioactivity) and sphingomyelin. 4. 4. Based on an assay of lipid phosphorus, dietary lecithin was found to be utilized by the larvae almost completely. The titre and specific activity of midgut lysolecithin suggest a role for this lipid in absorption.

An Assay Procedure for Inositol Pentaphosphate in Avian Erythrocytes

A method to determine the concentration of inositol pentaphosphate (IP5) in chicken erythrocytes is described. Lysates of washed erythrocytes were treated with trichloroacetic acid to remove protein and were chromatographed on Dowex-1 chloride columns. After eluting the other organic phosphates with .3 N HCl, IP5 was eluted with .5 N HCl and quantitated by phosphorus assay. The mean for 12 chickens was 15.4 ± 2.71 μmoles/g hemoglobin. The method should be useful for the assay of large numbers of samples.

A useful modification to the colorimetric assay of inorganic phosphorus and alkaline phosphatase in serum.

A useful modification to the colorimetric assay of inorganic phosphorus and alkaline phosphatase in serum.

Determination of phosphorus-containing compounds by spectrophotometry

No single method has been applicable for the determination of total phosphorus in the large spectrum of naturally-occurring and synthetic phosphorus-containing compounds, especially for those of extreme stability; e.g., ionic 0,O-dialkyl phosphates and 0-alkyl alkylphosphonates; the available phosphorus assay procedures determine only certain classes of phosphorus compounds. Phosphate esters from cells and tissues have been assayed routinely for total phosphorus after acid hydrolysis (e.g., 1) . Nonesterified alkylphosphonates have been determined after alkaline persulfate oxidation ( 2 ) ; however, 0-alkyl alkylphosphonates are not hydrolyzed quantitatively (3) , and the detection limit and the sensitivity in these determinations are poor (30-1600 nmol/assay). We have found (Cook and Daughton, unpublished data) that dry ashing with 5 M OB ( 4 ) results in incomplete and erratic recovery of dihydrogen 2-aminoethylphosphonate as orthophosphate. Subsequently, we found that a published method for wet ashing of aminoalkylphosphonates ( 5 ) could be extended and modified to give a simple, safe, general procedure for nongaseous phosphorus compounds in the 1-50 nmol range: this new procedure is described here. The orthophosphate resulting from the ashing was assayed by the highly sensitive method of Bartlett ( I ) .

Determination of disaturated lecithin in rhesus monkey amniotic fluid as an index of fetal lung maturity

Although increased concentrations of total lecithin in amniotic fluid allow prenatal assessment of fetal lung maturation, it has become clear that routine use of the L/S index may lead to a substantial number of inaccurate predictions. Since disaturated lecithin (DL) is a more specific marker of pulmonary surfactant than total lecithin, we developed a convenient method for measuring this phospholipid in amniotic fluid, and then evaluated its level in pregnant rhesus monkeys of 120 to 163 days of gestation. The method involves osmic acid destruction of unsaturated lipids, chromatographic isolation of disaturated lecithin, and quantitation by phosphorus assay. It can be performed in approximately 5 hours on 4 ml. of amniotic fluid and yields 67 ± 3 per cent average recovery of added 14C-dipalmitoyl lecithin. The results of analyzing 36 rhesus amniotic fluid specimens showed that disaturated lecithin and the disaturated lecithin/sphingomyelin ratio (DL/S) increase sharply after 150 days of gestation, consistent with the pattern of lung maturation in this species. We conclude that disaturated lecithin can be readily quantitated in primate amniotic fluid and that its concentration, the DL/S ratio, and percentage of disaturated lecithin are potentially useful indices of fetal lung maturity for the clinical laboratory.

Radioimmunoassay of pulmonary surface-active material in the tracheal fluid of the fetal lamb.

We measured pulmonary surfactant antigen by radioimmunoassay and phospholipid by lipid phosphorus assay in fluid collected during 12-hour periods from the trachea of lambs in utero between 102 and 147 days of gestation. Both phospholipid and antigen were detectable at 114 days and were present at low concentrations until 137 days. The concentrations then increased rapidly and reached 5- to 10-fold higher concentrations before parturition. The ratio of lipid phosphorus to antigen increased approximately 5-fold as the lungs passed from canalicular to alveolar architecture and approximated the adult ratio only during the alveolar stage of development.

Determination of inorganic phosphorus in the presence of organic phosphorus and high concentrations of proteins

The phosphorus assay method of Lowry and Lopez has been modified to permit a rapid and accurate determination of inorganic phosphorus in the presence of organic phosphorus without prior deproteinization of the samples. The reduction of ammonium phosphomolybdate to molybdenum blue is carried out with ascorbic acid in the presence of acetate buffer, pH 4.0, containing 10% sodium dodecyl sulfate to prevent protein precipitation. The method is applicable to samples containing up to 30 mg protein per milliter of final reaction volume. Acid-labile phosphorus compounds present in serum are not hydrolyzed, and less than 5% phosphocreatine is split during the time necessary for the assay. The addition of SDS to the buffer does not affect the sensitivity and reliability of Lowry and Lopez's method.

Assay of inorganic and organic phosphorus in the 0.1–5 nanomole range

Assay of inorganic and organic phosphorus in the 0.1–5 nanomole range

Effects of bilirubin, hemoglobin, and lipids on the Teepol and the SMA 12/60 methods for phosphorus

The phosphorus assay method of Lowry and Lopez has been modified to permit a rapid and accurate determination of inorganic phosphorus in the presence of organic phosphorus without prior deproteinization of the samples. The reduction of ammonium phosphomolybdate to molybdenum blue is carried out with ascorbic acid in the presence of acetate buffer, pH 4.0, containing 10% sodium dodecyl sulfate to prevent protein precipitation. The method is applicable to samples containing up to 30 mg protein per milliter of final reaction volume. Acid-labile phosphorus compounds present in serum are not hydrolyzed, and less than 5% phosphocreatine is split during the time necessary for the assay. The addition of SDS to the buffer does not affect the sensitivity and reliability of Lowry and Lopez's method.

Lipid composition of rat superior cervical ganglion.

AbstractExcised rat superior cervical ganglion were incubated in Krebs‐Ringer solution, freeze‐dried, and lipids extracted with chloroform: methanol (2∶1) v/v. Chromatography of lipid extracts in three separate thin layer chromatography solvents was accomplished on a single thin layer chromatography plate coated with acid‐washed Silica Gel H. Individual lipids were eluted from the silica gel using a modified Swinny filter holder technique and transesterified with methanolic‐BF3. Fatty acid methyl esters were separated and quantitated by gas liquid chromatography, while phospholipids were determined with a Malachite green dye organic phosphorus assay. Quantitation of neutral lipids was accomplished with a micro Liebermann‐Burchard technique and sphingolipids estimated colorimetrically as free sphingosine. The composition of lipids from freezedried rat ganglia resembles the lipid composition of rat brain. Phosphatidylcholine and phosphatidylethanolamine represent 76% of the glycerolphosphatides. The sphingolipids were comprised primarily of sphingomyelin with moderate levels of cerebroside and sulfatide. Cholesterol was the predominant neutral lipid. The major phospholipid fatty acids included palmitic, stearic, and oleic acids.

Polyphosphates and fish: some chemical studies

SummaryPolyphosphates are widely used for treating fish, but little information is available on the amounts taken up by fish and the suitability of some commercial polyphosphate mixtures. Phosphorus assays were performed on extracts of fish muscle which had been treated or not treated with polyphosphate mixtures. Fish‐to‐fish variation in natural phosphorus content was so large that it was difficult to determine whether or not fish had been treated with polyphosphate. The amount taken up under the conditions of treatment adopted was equivalent to 50–100 mg P/100 g fish muscle.Earlier experiments showed that sodium tripolyphosphate and sodium pyrophosphate were the most active condensed phosphates for preventing drip in prepacked chilled fish. The commercial mixtures found to give the best anti drip effect contained a high proportion of one or both of these two phosphates.

Influence of Dietary Fillers on the Performance of a Phosphorus Assay Diet

Two experiments using a total of 1260 broiler-type chicks were conducted to evaluate the use of diluents in phosphorus assays. Two formulations were used with either solka-floc or sand as a diluent and a third formulation had no diluent added. Although the diets without a diluent were more concentrated, the results obtained with them were not significantly higher than those with diluents. The diets containing sand resulted in the most uniform data. Solka-floc appeared to depress growth. Of the formulations studied, sand appears to be desirable in assay work with practical-type diets.

Addendum: A warning: An accurate and convenient organic phosphorus assay

Addendum: A warning: An accurate and convenient organic phosphorus assay

Improved Semiautomated Method for Assay of Phosphorus in Serum Phospholipids

Abstract A semiautomated method is described for assay of serum phospholipids, in which an AutoAnalyzer is used. This analytically accurate procedure eliminates difficulties in the automation mechanics of a previously reported method having the same chemical basis.

A Comparison of Phosphorus Assay Techniques with Chicks: 8. Evaluation of Two Dietary Levels of Plant Phosphorus for Use in Phosphorus Assays

EXTENSIVE evaluation has been conducted by many researchers to determine relative availability of both organic and inorganic phosphorus sources using several biological and chemical criteria. Plant phosphorus has been reported to be low in biological availability for the chick and the rat by Lowe et al. (1939), Krieger et al. (1940 (1941), Spitzer et al. (1948), Gillis et al. (1949 (1957), and Matterson et al. (1946). However, Waldroup et al. (1965) reported that optimum weight gains but not maximum mineralization could be obtained from a hominy feed diet which contained 0.5 percent phosphorus, all of which came from an organic source. These weight gains were superior to those obtained from a degerminated corn meal diet which had a total of 0.5 percent phosphorus with 0.2 percent from inorganic sources. The phosphorus supplemented degerminated corn diet in Waldroup’s trial was far superior to the all-plant phosphorus 0.5 percent levels for bone…

An accurate and convenient organic phosphorus assay

An accurate and convenient organic phosphorus assay

A Comparison of Phosphorus Assay Techniques with Chicks: 7. Comparison of the Relative Performance of Eight Phosphate Sources

SEVERAL studies have been conducted in an effort to establish the comparative utilization by chicks of phosphorus from various supplements. These reports were reviewed by Motzok et al. (1956) and Hurwitz (1964). In these studies the bilogical availability was derived by relating the percentage of tibia, beak, or toe ash of chicks fed a test phosphate to that of chicks fed a reference phosphate.In the past, considerable variation in the assay procedure employed has existed. One important variation has been in the calcium: phosphorus ratios or total calcium levels used in the experimental diets. Waldroup et al. (1965) reviewed certain experiments in which this was the case. In this paper it was pointed out that variations in the calcium content of the diet used in assaying phosphorus sources may significantly influence the relative biological value of a particular phosphate source. It was suggested that the use of a different…

A comparison of phosphorus assay techniques with chicks

Synopsis Two experiments were conducted to determine whether previously suggested calcium levels were adequate for use in assaying phosphorus availability from soft phosphate, defluorinated phosphate, and calcium phosphate. Results indicate that the levels were capable of producing maximum growth rate and maximum bone ash. The tibia ash data indicated that all sources of phosphorus were equal in biological availability. However, only defluorinated and calcium phosphate produced maximum growth. No explanation is given for the ability of soft phosphate to produce high bone mineralisation without maximum growth rate.