Total Protein Estimation Research Articles

Serum calcium measurement: Total versus free (ionized) calcium

Measurement of serum free (ionized) calcium (Ca(++)) reflects true calcium status of the body in health and disease. Present study evaluates efficacy of Ca(++) over total calcium (CaT) in serum for calcium status. 52 subjects were enrolled for study. Anaerobic fasting blood sample for Ca(++) measurement and autoclaved plain bulb for estimation of CaT, Total protein (TP) and Albumin was used. CaT, Corrected CaT, Ca(++), Calculated Ca(++) were measured and correlated. Corrected CaT and calculated Ca(++) were derived from the measured parameters. Study group showed significant difference between CaT and corrected CaT (p<0.006), Ca(++) and calculated Ca(++) (p<0.001). Negligible correlation was observed between Ca(++) and serum protein. Positive correlation was observed between CaT and calculated Ca(++), TP and albumin. Findings indicate that Ca(++) levels are independent of serum protein status. With scrupulous sampling, Ca(++) may be a better parameter than presently used CaT for assessing calcium status in serum.

Changes in serum levels of Total protein , Albumin and Globulin in patients with Behcet's Disease.

In order to have an idea about some of biochemical changes which might play a role in the pathogenesis of Behcet's disease. Estimation of serum total protein, albumin, globulin and HLA typing were carried out in 70 patients with Behcets disease using colorematric, mathematic &amp; microlymphocytotoxicity tests respectively, compared with 35 patients with recurrent oral ulcers, and 35 healthy control group.Hyperproteinemia, hyperglobulinemia with hypoalbuminemia were observed in those patients in comparison to control groups

Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells.

Submission of wild-type Escherichia coli to heat shock causes an aggregation of cellular proteins. The aggregates (S fraction) are separable from membrane fractions by ultracentrifugation in a sucrose density gradient. In contrast, no protein aggregation was detectable in an E. coli grpE280 mutant either by this technique or by electron microscopy. In search of an explanation for this observation at a molecular level, two kinds of marker proteins were used: Fda (fructose-1,6-biphosphate aldolase), the previously identified S fraction component, and IbpA/B, small heat-shock proteins abundantly associated with the S fraction proteins. Both types of marker proteins, normally never found in the outer-membrane (OM) fraction of WT cells, were present in the OM fraction from grpE cells after heat shock. This pointed to the presence of aggregates smaller than those in WT cells that cosedimented with the OM fraction. The OM fraction was enlarged in grpE cells. Although not proven directly, the presence of still smaller aggregates, not exceeding the solubility level and containing inactive Fda, was noted in the soluble CP fraction containing the cytoplasmic and periplasmic proteins. Therefore, aggregation occurred in both strains, but in a different way. The autoregulation of the heat-shock response causes a greater increase of DnaK/DnaJ and IbpAB levels in grpE cells than in WT after temperature elevation. This may explain the prevalence of the small-sized aggregates in the grpE cells. Estimation of total Fda protein before and after heat shock did not show any loss. This indicated that renaturation rather than proteolysis underlies the final disappearance of the aggregates. Though surprising at first, this is not contradictory with the participation of heat-shock proteases in removal of protein components of the S fraction as shown before, since proteins that are irreversibly denatured are probably substrates for the proteases.

Wilson's disease: the importance of measuring serum caeruloplasmin non-immunologically.

Wilson's disease should be considered as a possible diagnosis in any child, adolescent or young adult with liver damage without other explanation, especially when haemolysis is present. However, it may also present in adolescents or young adults with neurological signs confined to the motor system. The first diagnostic screening test is the estimation of the serum caeruloplasmin and total serum copper concentrations, with calculation of the serum non-caeruloplasmin-bound ('free') copper. Serum caeruloplasmin, which contains copper, is best determined by measurement of its oxidase activity, as the immunonephelometric method measures both caeruloplasmin and the biologically inactive apo-form. Diagnosis may be confirmed by an elevated urinary copper excretion. All close relatives of an identified patient must be screened and, where doubt persists, investigation of the Wilson's gene at chromosome 13q14.3 can be employed. Lifelong follow-up studies are best conducted in a specialist centre. Compliance with chelating therapy (penicillamine or trientine) or administration of the metal antagonist tetrathiomolybdate or zinc is monitored by determination of the serum 'free' copper, which should be maintained at or near 1.6 micromol/L (10 microg/100 mL). Side-effects of therapy are detected by the estimation of urinary total protein, full blood count and erythrocyte sedimentation rate, clotting factors and liver function tests.

Interference of Thiomersal in Biologicals During Protein Estimation

In the present report thiomersal was detected as interfering substance in hepatitis B vaccines during the total protein estimation by Lowry's protein assay. The thiomersal at different concentrations of 0·005%, 0·0075%, 0·01%, 0·02%, 0·05% and 0·1% was found to reduce the Folin Ciocalteu's phenol reagent and produce colour development between 0·024 O.D to 1·023 O.D. values. Further, the thiomersal was shown to interfere between 34·55% to 52·73% with Folin Ciocalteu's phenol reagent, when 10 batches of different hepatitis B vaccines were subjected to estimation of total protein content by Lowry's protein assay. Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.

Antiulcerogenic activity of Lippia alba (Mill.) N. E. Brown (Verbenaceae)

Lippia alba (Mill.) N. E. Brown Verbenaceae, known popularly as ‘Juanilama’ or ‘Salvia Sija’, is prized widely in folk medicine in Guatemala. Its leaves are employed as an infusion and decoction as a remedy for stomach problems, dysentery, colds and cough, febrifuge, as well as a sedative and in spasmolitic remedies. The present study reports the effects of the infusion of L. alba on the rat gastric mucosa. The following behavioural parameters were evaluated: (a) gastric irritancy test in Wistar rats; (b) antiulcer activity, short term and long term; (c) acid secretion; (d) measurement of total proteins; (e) estimation of total protein bound and nonprotein sulfhydryl groups. Ranitidine (100 mg/kg, p.o.) was used as the reference antiulcer drug. Oral treatment with the infusion (12.5 g dry plant/kg) did not cause gastric irritancy in the rats treated during five consecutive days. In addition, the oral administration of L. alba was found to be effective at preventing gastric ulceration induced by indomethacin (50 mg/kg, p.o.) in rats in the short term (1 day) and long term (5 days).

Hepatocyte growth factor in human semen and its association with semen parameters.

Hepatocyte growth factor (HGF) is a structurally unique growth factor with potent motogenic (motility inducing) effects. Studies in the murine male genital tract have suggested important associations between HGF and the acquisition of sperm motility during epididymal maturation. The aim of this study was, therefore, to determine the concentration of HGF in human semen and assess its correlation, if any, with sperm motility and other semen parameters. Semen samples were collected by masturbation and analysed using standard procedures. HGF concentrations were measured in duplicate using an enzyme-linked immunoassay technique. Total protein estimations were also made in a subset of samples. The 95 subjects were divided into three groups for analysis: normozoospermic, subnormal semen and azoospermic. HGF was detected in all samples (median 0.456, 25th centile 0.388, 75th centile 0.556 ng/ml). No significant correlations were found between semen HGF concentrations and sperm concentration, motility, total sperm count or total motile count. There were no significant differences in mean HGF concentrations between the three subgroups. In conclusion HGF is present in human semen in significant quantities. The data do not suggest HGF concentrations are correlated with parameters of sperm motility.

An on-line method for the measurement of total protein output in biological fluids and secretory tissues after stimulation of intrinsic nerves and identification of neurotransmitters using immunohistochemical techniques

Proteins are essential ingredients of life and thus it is essential to measure the level of proteins in biological fluids and tissue homogenates. Several methods have previously been described in the literature for the estimation of proteins using either certain dyes which bind to specific groups of polypeptide side chains producing a protein dye colour complex, methods involving copper binding to peptide bonds or application of an eosin B red dye. In this study, an on-line automated technique based on the Lowry method has been used to estimate total protein output from the isolated lacrimal segments. This method can also be used to estimate total protein from saliva, or any other biological fluid, tissue homogenates or secretory tissues. The on-line automated method for the estimation of total protein from secretory tissues and biological fluid was designed mainly to obtain a rapid, simple and consistent graphical interpretation of result within 40–50 min of starting the experiment. The original chart recording of the time-course response can also be used for publication purposes. With this method, it is possible to investigate the effect of electrical field stimulation on the intrinsic secretomotor nerves employing either wire or platinum electrodes embedded in the perfusing chamber. Moreover, the tissue can also be stimulated with different concentrations of either drugs, hormones or neurotransmitters for different time periods. This method can also be combined with morphology whereby the stimulated tissue can be processed for neuropeptide or neurotransmitter immunohistochemistry to determine which neurotransmitters or neuropeptides are involved in the physiological responses. The automated method is simple and rapid and moreover, it can estimate accurately and directly at physiological pH small amounts (ng–μg) of proteins in effluent samples depending on the sensitivity of the chart recorder. In this study, neuropeptides and neurotransmitters were used as secretagogues in addition to electrical field stimulation. Themes: Neurotransmitters, modulators, transporters, and receptors Topics: Adrenaline, acetylcholine, peptides and anatomy and physiology

Investigating paediatric airways by non‐bronchoscopic lavage: normal cellular data

Bronchoscopic bronchoalveolar lavage in children to investigate bronchial disorders such as asthma has both ethical and procedural difficulties. The aim of this study was to establish a standardized non-bronchoscopic method to perform bronchoalveolar lavage in children attending for elective surgery to obtain normal cellular data. Bronchoalveolar lavage was performed on normal children (n = 55) by infusing saline (20 mL) through an 8 FG suction catheter passed after endotracheal intubation. Oxygen saturation, heart and respiratory rate were monitored during the bronchoalveolar lavage procedure. Cellular analysis and total protein estimation of the lavage fluid were performed. Epithelial lining fluid volume was calculated (n = 15) using the urea dilution method. The procedure was well tolerated by all children. Total cell count and differential cell count for children (macrophages 70.8 +/- 2.3%, lymphocytes 3.8 +/- 0.6%, neutrophils 5.7 +/- 1.0%, eosinophils 0.14 +/- 0.03%, epithelial cells 19.6 +/- 2.1%, mast cells 0.21 +/- 0.02%) were similar to those reported for adults. Age and sex comparisons revealed no differences between groups. The mean total protein recovered in the cell free supernatant was 49.72 +/- 4.29 mg/L and epithelial lining fluid volume was 0.82 +/- 0.11% of return lavageate. This method allows bronchoalveolar lavage to be performed safely and quickly on children attending for routine elective surgery. Using this method and taking the 'window of opportunity' of elective surgery, the presence or absence of airway inflammation could be studied in children with various patterns of asthma during relatively asymptomatic periods.

Misuse of the Kjeldahl Method for Estimating Protein Content in Plant Tissue

Accurate plant protein analysis is difficult, primarily because of the small amounts of proteins present in the tissue and partly due to the interference of other complex plant constituents (Agusti and Beltran, 1982; Khanizadeh et al., 1989, 1992a; Marks et al., 1985; Robinson, 1979). Many procedures have been used for protein determination in plant tissues. The most commonly used procedures for total protein estimation are: folin phenol (Lowry et al., 1951), biuret (Gornall and Bardawill, 1949), 280/260 nm ultraviolet (UV) absorption, improved 224–236 nm isoabsorbance (Groves et al., 1968), 280/205 nm absorption (Scopes, 1974), coomassie blue dye binding (Bradford, 1976), and classical Kjeldahl [Association of Official Analytical Chemists (AOAC), 1984; Morries, 1983]. However, some of these methods (e.g., folin phenol, Bradford and UV absorption, and biuret) cannot be used for plant tissue due to the presence of numerous interfering compounds, such as phenolics, tannins, and alkaloids (Benedict, 1987; Marks et al., 1985; Peterson, 1983; Robinson, 1979). Others, such as the spectrophotometric method of Warburg and Christian (1942) and Waddell and Hill (1956), cannot distinguish between protein and plant nucleic acid content (Groves et al., 1968). The biuret reaction is also subject to ammonia, tris, and glycerol interference (Bradford, 1976). Peterson (1983) examined various methods of protein determination based on their relative sensitivity for bovine serum albumin and a range of proteins. He concluded

Quantitation of monoclonal antibodies by ELISA: The use of purified mouse IgG and mouse IgM monoclonal antibodies as standards in a quantitative ELISA measuring monoclonal antibodies produced by cell culture

Murine monoclonal antibodies (MAbs) of IgG1, IgG2a and IgG2b subclasses and IgM class in hybridoma culture supernatants were quantified using a sensitive, reliable, optimized indirect double antibody sandwich ELISA. In the ELISA, the MAb in the culture supernatants was sandwiched between affinity isolated heavy chain specific polyclonal antibodies used for capture and detection. Quantitation was achieved by comparison with a standard curve produced by a purified MAb of the same class, subclass or ideally the same clone as the MAb to be quantified. These quantitative results were compared with those obtained using purified IgG and IgM polyclonal serum samples as standards and those obtained by total protein estimation using measurement at OD 280 nm. The IgG subclass MAbs used as standards were purified using protein G and the IgM class MAb was purified by ion exchange followed by gel filtration chromatography. Bovine IgG contamination of the MAb supernatants and the purified MAbs was also measured by a double antibody sandwich ELISA.

External Quality Assessment of Total Urinary Protein Estimation in the United Kingdom

Three surveys of total urinary protein quantitation have been carried out in 350 UK laboratories. The seven specimens comprised buffered saline or normal human urine with added human serum albumin or human serum, or urine from individuals with nephrotic syndrome. Principal method groups were: turbidimetry (57%), dye binding (25%) and biuret (15%). For all surveys, overall between-laboratory agreement was poor (CV 22.8% to 57.1%), with ranges of results from 24-fold (0.83-20 g/L) to 366-fold (0.05-18.3 g/L); there was no improvement with time. The most popular method (sulphosalicylic acid turbidimetry) consistently performed the worst, and performance of the direct biuret procedure was also unacceptable; both methods should be discontinued. There were no significant differences in performance between the other major method groups, and none can be specifically recommended. Within the individual calibrant groups, least variation was observed with human serum. A common calibrant for all participants yielded significantly better between-laboratory agreement for all methods except sulphosalicylic acid turbidimetry.

Experimental selenium toxicity in guinea pigs: biochemical studies.

Biochemical studies were conducted in experimentally induced selenium toxicity in recently weaned guinea pigs. A significant drop in blood glucose level, in comparison to controls, was observed in animals fed selenium-enriched barley (organic form) as well as those fed ordinary barley mixed with sodium selenite (inorganic form). Estimation of total serum proteins also revealed a significant drop in both these groups. SGOT (EC 2.6.1.1) activity was comparatively lower but no significant alteration was noticed in SGPT (EC 2.6.1.2). The erythrocytic glutathione peroxidase activity was significantly increased in inorganic selenosis followed by that in organic one, in comparison to controls. All these alterations were of mild degree in guinea pigs which were given sodium arsenite (10 ppm) along with sodium selenite (30 ppm) in the feed.

Urinary total protein estimation--fact or fiction?

In two recent surveys of urinary total protein assays, 370 laboratories in the United Kingdom were requested to determine the protein content of a simulated 24-hour urine (a solution of sodium and potassium salts and urea, with no added protein) and of a 24-hour urine from a healthy individual. The nature of these specimens was not revealed and participants used their routine methods and calibrants. Quantitative results (range 0.005-12.23 g/l, median 0.03 g/l) were received from 31% of the participants for the salt solution and from 43% for the normal urine (range 0.01-2.96 g/l, median 0.05 g/l). Nonquantitative results, i.e. those given as less than a detection limit (range less than 0.005 to less than 0.5 g/l) were received from 29% of the participants for the salt solution and from 33% for the normal urine. Statements of 'nil', 'zero' or 'not detected' were received from the remainder. Further analysis of the results indicated that 29% of the laboratories reported, or did not unequivocally exclude, significant proteinuria in the salt solution, and 41% of the laboratories similarly did not exclude proteinuria in the normal urine. It is proposed, for both clinical and analytical reasons, that consideration be given to the discontinuation of urinary total protein estimation and that urinary albumin, supplemented where appropriate by other selected protein or enzyme measurements, be determined instead.

Immunohistochemical Micromethods for the Measurement of Specific Collagen Types in Human Liver Biopsies

Micromethods are needed for measuring the amount of collagen and other matricial proteins in human liver biopsies. Recently, a colorimetric method for the quantitative estimation of total collagen and protein has been developed. This method is sensitive, can be applied to fresh and fixed tissues and provides the absolute value of total collagen per section. Results are expressed as microgram of collagen per mg of proteins or per section. We now report two micromethods for the determination of specific collagen types in fresh frozen liver sections. The first method is a direct method utilizing the tissue section as an ELISA plate. Results are expressed as the amount of color reaction read at 450 nm per slide. After cleaving the antigen antibody complex with dilute acetic acid, the amount of total collagen can be measured by the colorimetric microchemical method and the amount of specific collagen type is expressed as the amount of color reaction per microgram of total collagen. The second method is an indirect method: the floating tissue section is incubated with an optimal amount of specific anti-collagen antibody and the residual amount of antibody present in the supernatant is measured in an ELISA plate coated with the specific collagen type. The amount of specific collagen type present in the tissue section is determined by comparing the optical deviation obtained with a standard curve prepared with known amounts of specific collagen, and values are expressed as microgram of collagen per tissue sections. As with the direct method, the amount of total collagen per section can be measured by the colorimetric method and then, the amount of specific collagen type can be expressed as microgram of specific collagen type per microgram of total collagen.

A Comparison of the Glutaraldehyde Coagulation Test and Total Serum Protein Estimation as Indicators of Gamma Globulin Levels in Neonatal Ruminants

A Comparison of the Glutaraldehyde Coagulation Test and Total Serum Protein Estimation as Indicators of Gamma Globulin Levels in Neonatal Ruminants

The effect of road transportation on the blood constituents and behaviour of calves. II. One to three weeks old

Six calves, one to three weeks old, were transported by road for 6 h (group J) and another six for 18 h (group K). Blood samples taken before, during and after the journeys were analysed for haematocrit, total and differential leucocyte count, plasma corticosteroid and glucose, serum non-esterified fatty acids (NEFA), total protein and calcium levels, aspartate amino transferase (AST) and creatine kinase (CK) activities. In comparison with the levels found in a group of six calves only starved for 18 h (group L) there were no significant changes in haematocrit, total leucocyte count, serum NEFA, total protein, calcium, AST and CK estimations of transported calves (group J & K). The plasma corticosteroid concentration rose significantly only after loading and, in group K, immediately after unloading. A decrease in plasma glucose occurred after the 6-h journey when calves were not fed for a further 12 h. There was a significant increase in neutrophil numbers and decrease in lymphocyte numbers in both transported groups of calves. The mean loss of weight resulting from 6-h transportation was 1·95 kg (1·5 kg starvation only) and for 18 h 2·95 kg (2·36 kg). Calves lay down for 0 to 55% of the journey and ruminated for 2·9 to 35% of the time.

Effect of subdermal levonorgestrel contraceptive implants, NORPLANT ®, on liver functions

Forty-seven normal health women were studied longitudinally for changes in liver functions during the use of the levonorgestrel contraceptive implant system, NORPLANT ®. Samples were collected before insertion of the implants and after one, three and six months of use. The enzymes studied were the transaminases (SGOT and SGPT), alkaline phosphatase and -glutamyl transferase. Serum bilirubin and bile acid levels were also measured. The protein synthetic function of the liver was tested by estimation of total proteins, albumin, transferrin, hemopexin, ceruloplasmin and haptoglobin. The three main immunoglobuins, G, M and A, were also measured. There were no significant changes in the liver enzymes after NORPLANT ® use. Serum bilirubin and bile acid concentrations showed rises in the first month of use which ameliorated in subsequent months. Serum albumin was transiently increased during the first and third months. Ceruloplasmin decreased significantly at the sixth month. The concentrations of total serum proteins and the other individual proteins showed no significant change. The results point to safety of NORPLANT ® implant use, as regards hepatic functions.

Estimation of Soluble Protein in Must and Wine by High-Performance Liquid Chromatography

Estimation of total soluble protein in must and wine by high-performance liquid chromatography is achieved with a size exclusion column. A protein standard, bovine serum albumin, is eluted from this column with linear response to a concentration of 1 g/L. Protein is separated from other UV absorbing components of must and wine and estimated at 280 nm on a Waters Protein Separation System with a 0.1-M ammonium acetate mobile phase containing 10% glycerol. The method is now used routinely for estimation of protein in must and wine.

Prolactin in human milk: correlation with lactose, total protein, and alpha-lactalbumin levels.

To determine whether prolactin (PRL) could be conclusively demonstrated in human milk, samples from nine puerperal women were examined by radioimmunoassay techniques. Recovery of 78 +/- 5% (mean +/- SE) PRL added to eight milk samples and mean recoveries of 83, 92, and 92% of PRL after 1, 2, and 3 h incubation at 21 degrees C indicated that quantitative recovery was possible. PRL immunoreactivity was not lost by dialysis for 96 h. Serial dilution of milk samples before and after dialysis gave inhibition curves parallel to PRL standards. Milk PRL concentrations were high for the first 3 days after birth, with a peak mean value of 157 +/- 18 ng/ml (SE) on the third day. Milk PRL levels then fell sharply to a mean of 24 ng/ml 13 days after delivery. Milk PRL concentrations showed a significant negative correlation (P less than 0.001) with milk lactose (r = -0.59) and positive correlations (P less than 0.01) with total milk protein (r = 0.49) and alpha lactalbumin (r = 0.33) estimations. We conclude that i) PRL is a normal constituent of human milk, ii) high concentrations of PRL are present in human milk for the first 3 days postpartum but subsequently fall rapidly, and iii) milk PRL levels correlate significantly with milk lactose, total protein, and alpha-lactalbumin values.