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PubMed1989

Performance profiles: new tools for characterization and comparison of clinical chemical results.

It is current practice to record the performance of immunoassays by means of precision profiles (according to Ekins), in which the variation coefficient (relative standard deviation) is plotted against the analyte concentration. On the other hand, precision profiles are only occasionally used for evaluating the performance of conventional clinical-chemical methods. It is relatively uncommon to find bias plotted against analyte concentration, presumably because this type of analysis requires reference specimens, whose true analyte concentrations are known or guaranteed by reference methods. If the relative systematic variations are plotted against the true analyte concentrations, and a confidence interval is added to the resulting regression curve, the result is a "bias profile"; if tolerance limits are added, the result is a "deviation profile". The present work describes the preparation of specimens, which can be used to provide experimental data for the three performance profiles. A computer program is used to construct the precision profile, bias profile and/or deviation profile. The mathematical-statistical basis of the program is described in detail. For evaluation of the statistical procedure, three analytes and six methods were used: determination of sodium activity/concentration with an ion sensitive electrode and by flame photometry; determination of creatinine by a manual enzymic and a mechanized Jaffé method; determination of thyrotropin by radioimmunoassay and by luminescence immunoassay. Different purposes are served by bias and deviation profiles. Thus, bias profiles can be used to compare the bias of two methods, whereas a deviation profile can be used to define the analytical range of a method. If the acceptable limits of deviation are added to the deviation profile, then the useful analytical range of the method is immediately apparent.

Chemistry, Clinical↗
PubMed1994

A manual spectrophotometric method for the measurement of serum sodium and potassium by enzyme activation.

Manual procedures suitable for use on standard benchtop spectrophotometers have been developed for the enzymatic determination of Na+ and K+ in serum. Both assays require only minimal modification of reagents already available for BM/Hitachi analyzers and are performed in an endpoint mode, allowing up to 20 assays per run. The addition of a stop reagent is required--dipotassium EDTA for the Na+ assay and sodium dodecyl sulphate for the K+ assay. The most important criterion for achieving good assay performance is the precise pipetting of sample and reagent. Within-run imprecision is < 1% for Na+ and K+, and between-run imprecision < 1.5%, for both assays at all but the lowest concentrations of K+. Enzymatic electrolyte results compare well with flame photometry, however the assays are more prone to interference by very high concentrations of bilirubin or triacylglycerols than those performed on automated, dual-wavelength kinetic analyzers. It is possible to correct for most interferences by inclusion of appropriate sample and reagent blanks.

Absorption↗
PubMed2004

Evaluation of a new lithium colorimetric assay performed on the dade behring dimension X-pand system.

Lithium is the standard first line of treatment for bipolar disorder. Because of its potential toxicity, levels of the drug have to be constantly monitored. To this end, an accurate and rapid assay is thus required. Moreover, lithium measurement allows differential diagnosis in patients with hallucinations, dementia and amnesia. The lithium concentration in blood is usually evaluated by flame photometry, atomic absorption spectroscopy or ion selective electrode. The aim of this study was to evaluate the analytical performance of a new colorimetric assay for lithium. Within-day precision of a pool with a target value of 1.25 mmol/l was 2.18% (CV) and day-to-day precision was assessed using serum aliquots containing two concentrations of lithium, and CVs were 5.47 and 1.6% at 0.6 and 1.15 mmol/l, respectively. The lower detection limit (LLD) was <0.08 mmol/l. The assay seemed to be linear up to 5 mmol/l. Results of a comparison method using Deming regression were: y=1.161x-0.0075 (r=0.9879) for regression with the Flame Photometer and y=0.9729 x+0.0133 (r=0.9787) for regression with atomic absorption spectroscopy. Moreover, in this study we did not find any interference with drugs or other analytes tested. In conclusion, this assay may be a method of choice for measuring lithium blood concentrations, especially in emergency situations.

Antimanic Agents↗
PubMed1988

Opposing effects of glucose and tolbutamide on the sodium content of rat pancreatic islets.

Integrating flame photometry was employed for measuring sodium in rat pancreatic islets incubated in media buffered with HEPES or bicarbonate. The sodium content decreased by nearly 40% when the islets were exposed to 5 mmol/l glucose, no further reduction being seen with additional rise of the concentration to 20 mmol/l. Whereas the depressing effect of glucose was mimicked by 100 mumol/l quinine, increased sodium contents were noted after inhibition of the Na/K pump (removal of extracellular K+ or addition of 1 mmol/l ouabain) or exposure of the islets to 1 mmol/l tolbutamide. Although promoting sodium accumulation in the islet cells, tolbutamide counteracted the increase in sodium obtained on withdrawal of K+ from the incubation medium. It is suggested that tolbutamide in addition to its major effect in promoting the entry of Ca2+ also facilitates insulin release by suppressing the outward transport of this ion.

Animals↗
PubMed2002

Evaluation of flow injection analysis for determination of urea in sheep's and cow's milk.

Difficulties in measuring the urea content in sheep's milk often occur with spectral photometry due to the high protein and fat concentrations of the milk. In this study an enzymatic flow procedure (QuickChem 8000 Ion Analyser, Lachat Instruments, Milwaukee, USA) to determine the urea content in ovine and bovine milk was evaluated. Urea content is determined by the Berthelot reaction after splitting it enzymatically with urease. The free ammonia diffuses through a teflon membrane into a stream of reagent solutions. Detection takes place by means of a reaction between the ammonium ions with hypochlorite and salicylate producing a green colour, which is measured spectrometrically in a flow meter at 660 nm. By using a diffusion cell chemical deproteinisation of milk is not necessary and capacity is high. The assessed procedure exhibited high accuracy and precision and reached a sample capacity of 55 samples an hour. Storage of the milk samples for several days as well as chemical preservation with bronopol had no effect on the measurement procedure. Due to the complexity of the apparatus and the costs associated therewith, the device proves less suitable for routine diagnostics but rather serves as a reference method for the measurement of urea concentration in milk.

Animals↗
PubMedDate not supplied

Polyunsaturated but not conjugated linoleic acid supplementation of leukemic U937 cells can act as an amplification factor for photofrin-mediated photodynamic therapy.

Polyunsaturated fatty acids located in leukemia cell membranes are excellent targets for peroxidation. They can significantly enhance the effectiveness of Photofrin-mediated photodynamic therapy (PDT)-induced cell killing. In this study, the peroxidizability of conjugated fatty acid isomers (9c,11t-linoleic acid and 9c,11c-linoleic acid) and polyunsaturated fatty acids (PUFAs; linoleic acid, gamma-linolenic acid and arachidonic acid) with 2,2'-azo-bis(2-amidinpropane)dihydrochloride, soybean lipoxygenase and photomediated peroxidation are compared with each other. Peroxidation was determined using different methods: by means of gas chromatography to estimate the fatty acid (FA) consumption, by photometry for the level of FA peroxides or phospholipid peroxides and by definition of the content of malondialdehyde for thiobarbituric acid reactive substances (TBARS). The results suggest that the generation of oxidation products from individual FAs indicate a different formation rate of oxidation products. Radical FA peroxides were produced most by polyunsaturated arachidonic acid, followed by linoleic acid and gamma-linolenic acid, whereas conjugated FA isomers did not generate peroxides. Accordingly, the levels of lipid peroxides and TBARS were substantially increased after incorporation and oxidation of polyunsaturated FAs into U937 cells and could significantly enhance the effectiveness of Photofrin-PDT-induced cytotoxicity. The results showed that PUFA, but not conjugated FA supplementation of U937 cells, can act as a PDT amplification factor.

Cell Line↗
PubMed1997

Agonist-induced calcium regulation in freshly isolated renal microvascular smooth muscle cells.

The studies presented here were performed to determine the effect of agonist stimulation on the cytosolic free Ca2+ concentration ([Ca2+]i) in single smooth muscle cells, freshly isolated from afferent arterioles and interlobular arteries averaging between 10 to 40 microns in diameter. Microvessels were obtained from male Sprague-Dawley rats using an iron oxide collection technique followed by collagenase digestion. Freshly isolated microvascular smooth muscle cells (MVSMC) were loaded with fura 2 and studied using fluorescence photometry techniques. The resting [Ca2+]i averaged 67 +/- 3 nM (N = 82 cells). Increasing the extracellular K+ concentration significantly increased [Ca2+]i dose-dependently (P < 0.05). Involvement of extracellular Ca2+ in the response to KCl-induced depolarization was also evaluated. Resting [Ca2+]i increased approximately 132% from 40 +/- 5 nM to 93 +/- 26 nM in response to 90 mM extracellular KCl. This change was abolished in nominally Ca(2+)-free conditions and markedly attenuated by diltiazem. Inhibition of K+ channels with charybdotoxin or tetraethylammonium chloride produced a modest transient increase in [Ca2+]i during the response to 30 mM K+ and had no detectable effect on responses to 90 mM K+. Studies were also performed to establish whether freshly isolated renal MVSMC exhibit appropriate responses to receptor-dependent physiological agonists. Angiotensin II (100 nM) increased cell Ca2+ from 97 +/- 10 nM to 265 +/- 47 nM (N = 12 cells). Similarly, 100 microM ATP increased MVSMC [Ca2+]i from a control level of 71 +/- 14 nM to 251 +/- 47 nM (N = 11 cells). Norepinephrine administration caused [Ca2+]i to increase from 63 +/- 4 nM to 212 +/- 47 nM (N = six cells), and vasopressin increased [Ca2+]i from 86 +/- 10 nM to 352 +/- 79 nM (N = five cells). These data demonstrate that receptor-dependent and -independent vasoconstrictor agonists increase [Ca2+]i in MVSMC, freshly isolated from rat preglomerular vessels. Furthermore, the ability to measure [Ca2+]i in responses to physiological stimuli in these single cells permits investigation of signal transduction mechanisms involved in regulating renal microvascular resistance.

Adenosine Triphosphate↗
PubMed1993

Cloning and characterization of human gingival and periodontal ligament fibroblasts.

Fibroblast heterogeneity in human periodontal tissues was characterized by cloning and immuno-histochemical techniques. Cell suspensions from primary cultures gingival (GF) and PDL fibroblasts (PF) were cloned. The relative intensity of double-labeled immunofluorescence, using specific antibodies to the extracellular matrix (ECM) molecules collagen type I (CI), type III (CIII), and fibronectin (Fn), was measured by photometry. Most clones derived from either GF or PF showed positive intracellular staining for both CI and Fn, and CIII and Fn. However, there were variations in fluorescence intensity for CI and Fn, ranging from relatively weak to strongly positive. The fluorescence for CI and CIII was relatively weak in most isolated GF clones in contrast to their PF clones. These observations coupled with studies of growth and cellular morphology in individual clones suggest that: 1) GF and PF contain functionally heterogeneous subpopulations; and 2) the synthesis and expression of extracellular matrix molecules of GF may be essentially different from that of PF.

Clone Cells↗
PubMed1984

Evaluation of plasma histamine in patients with coronary artery disease.

Plasma levels of histamine (HI) were determined by fluorescence photometry in 12 patients with old myocardial infarction, 10 patients with stable effort angina pectoris and 10 control subjects without coronary arterial lesion. Resting HI were 57.6 +/- 7.5 ng/ml for control subjects and 68.6 +/- 3.9 ng/ml for patients with coronary artery disease (CAD) (N.S.). HI were 62.6 +/- 4.7 ng/ml for patients with one vessel disease, 68.0 +/- 10.6 ng/ml for patients with two vessel disease and 79.1 +/- 4.9 ng/ml for patients with three vessel disease (one vessel disease: three vessel disease, p less than 0.05). HI for all subjects decreased from 65.6 +/- 3.5 ng/ml before treadmill exercise test to 63.4 +/- 3.8 ng/ml after exercise (N.S.), and the rate of change did not differ from control subjects to diseased patients. HI increased significantly from 63.5 +/- 3.7 ng/ml to 70.7 +/- 3.7 ng/ml after hyperventilation (p less than 0.05), but the rate of change did not differ from control to CAD patients. In the present study a tendency was observed that the severe the coronary arterial lesions, the higher were HI. In old myocardial infarction and stable effort angina pectoris, however, the changes in HI produced by exercise and hyperventilation do not seem to be deeply involved in the onset of acute myocardial ischemia.

Coronary Disease↗
PubMed2003

Dynamics of calcium clearance in mouse pancreatic beta-cells.

Pancreatic beta-cells maintain glucose homeostasis by their regulated Ca(2+)-dependent secretion of insulin. Several cellular mechanisms control intracellular Ca(2+) levels, but their relative significance in mouse beta-cells is not fully known. We used photometry to measure the dynamics of cytosolic Ca(2+) ([Ca(2+)](i)) clearance after brief, depolarization-induced Ca(2+) entry. Treatment with thapsigargin or cyclopiazonic acid, inhibitors of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pumps, nearly doubled the peak and slowed the decay of the depolarization-induced Ca(2+) transients. The remaining thapsigargin-insensitive decay was slowed further by inhibition of the plasma membrane Ca(2+)-ATPase (PMCA) and plasma membrane Na(+)/Ca(2+) exchanger (NCX) via alkalization of the bath solution, by adding lanthanum, or by substitution of Na(+) with Li(+). Mitochondrial Ca(2+) uptake contributed little to clearance in thapsigargin-pretreated cells. Together, the SERCA, PMCA, and NCX transport mechanisms accounted for 89 to 97% of clearance in normal solutions. We developed a quantitative model for the dynamic role of removal mechanisms over a wide range of [Ca(2+)](i). According to our model, 50 to 64% of initial Ca(2+) removal is via the SERCA pump, whereas the NCX contributes 21-30% of the extrusion at high [Ca(2+)](i), and the PMCA contributes 21-27% at low [Ca(2+)](i).

Animals↗
PubMed1997

Sebum and water content in the skin of aged immobilized patients.

In the present study we investigated the sebum content and hydration of the skin in aged immobilized patients. Healthy aged as well as young and aged immobilized patients were evaluated by photometry, using a sebum tester and capacitance meter to detect sebum and hydration, respectively, in various skin areas. Sebum content was significantly higher in the young groups as compared to the aged ones, including those of the immobilized patients. Similar values of water content were observed in the healthy young and aged volunteers. However, surprisingly, a significantly marked decrease was detected in each tested area of the immobilized patients. We may assume that a cascade of events caused by the immobilization status of the patients leads to a significant decrease in water content within the dermis.

Adult↗
PubMed1998

Deconditioning reduces mineral content of the third metacarpal bone in horses.

Diet and exercise are two management factors that affect bone density and strength. We proposed that bone density and calcium status would be affected by deconditioning for 12 wk and by dietary Ca concentration. Eleven highly conditioned Arabian horses were taken out of training and placed in stalls for 12 wk. Horses were walked on a mechanical walker in two 30-min sessions, 7 d/wk. Diets were designated CC (.36% Ca) and HC (.62% Ca). Data were collected every 21 d. Serum or plasma were analyzed for total and ionized Ca, parathyroid hormone, osteocalcin, hydroxyproline, electrolytes, and blood gases. Bone mineral content (BMC) of the left third metacarpal bone was estimated by radiographic photometry using an aluminum step wedge, which was exposed in each radiograph, as a reference standard for an image analysis system. During deconditioning, BMC decreased by approximately 1.1 g/2 cm, or .45% per week. This decrease was unaffected by dietary Ca. Serum Ca concentration increased with deconditioning. The results suggest that dietary Ca at twice the currently recommended level did not prevent the loss of BMC in response to deconditioning. Loss of BMC during 12 wk of stall confinement may weaken bones, increasing the risk of skeletal injuries when training is resumed.

Absorptiometry, Photon↗
PubMedDate not supplied

Effects of intravenous glycerol injection on inner ear fluid electrolytes.

Under sodium pentrobarbital anesthesia (20-30 mg/kg, i.p.), normal guinea pigs received an intravenous injection of glycerol (1.0 ml/kg). Serum, cerebrospinal fluids (CSF) and inner ear fluids were collected from the scala tympani perilymph, scala vestibuli perilymph and the scala media endolymph. The sodium and potassium concentrations were assessed using microflame photometry. Increases in sodium concentration were found in the CSF and the scala tympani perilymph; no significant changes were observed in the serum, scala vestibuli perilymph or the scala media endolymph. These sodium increases were considered to be a result of the dehydration process caused by the osmotic agent glycerol. Increases in potassium concentration were found only in the scala vestibuli perilymph.

Animals↗
PubMed1988

Effect of cooling on the intracellular concentrations of Na+, K+ and Cl- in cultured human endothelial cells.

Cooling is accepted as a practical way of lowering cell metabolism in vein grafts during coronary by-pass surgery. We have previously shown that low temperature causes endothelial cells to become detached, both in in vitro and in vein graft. In this study we have looked at the effect of cold on the concentrations of intra- and extracellular electrolytes. Human endothelial cells were grown on titanium grids for electron microscopy. The cells were incubated for 30 min at 37 degrees, 20 degrees, and 4 degrees C with cell culture medium containing human serum, and at 20 degrees and 4 degrees C with heparinized sodium acetate solution with serum, frequently used for flushing and distending vein grafts. Freeze-dried cells were then subjected to elemental X-ray microanalysis. The ambient fluid was analysed by flame photometry. At 20 degrees and 4 degrees C, intracellular concentration of sodium increased, and potassium decreased, compared with controls (37 degrees C). The changes in sodium concentrations were aggravated when cell culture medium was replaced by heparinized sodium acetate. The intracellular chloride concentration did not change when cells were stored in cold cell culture medium. The extracellular concentration of potassium increased with increasing incubation time at 4 degrees C. The connection between these findings and cell detachment is discussed.

Chlorides↗
PubMed1995

Implantable biodegradable polymeric device in the treatment of experimental proliferative vitreoretinopathy.

We investigated the use of a scleral plug of biodegradable polymer implanted at the pars plana to create a controlled drug-delivery system in the vitreous. We evaluated the efficacy of a plug containing doxorubicin hydrochloride to treat experimental proliferative vitreoretinopathy (PVR) in pigmented rabbits. An implantable device on the sclera, which imitates a scleral plug, containing 1% doxorubicin, was prepared with poly(lactic acid) (molecular weight, 20,000). The release of doxorubicin in phosphate-buffered saline was evaluated by spectro-photometry. After pars plana vitrectomy and plug implantation, concentrations of doxorubicin in the vitreous humor of the rabbits were measured by high performance liquid chromatography. The release profiles were evaluated during 5 weeks in vitro and 4 weeks in vivo. Cultured homologous fibroblasts were injected into the vitreous space to induce experimental PVR after gas compression of the vitreous. The scleral plugs were implanted at the pars plana in treatment animals (n = 11). Control rabbits (n = 11) were followed up without implantation after PVR induction. All eyes of the control group developed tractional retinal detachment at day 28, while the incidence of retinal detachment was decreased to 64% in the treated eyes. (P = 0.002). The implantation of the scleral plug effectively inhibited intravitreous proliferation of fibroblasts. This study demonstrated that the scleral plug of biodegradable polymers may have potential as a treatment modality for PVR.

Animals↗
PubMed1989

Influence of thyroid hormone on myosin heavy chain mRNA and other messenger RNAs in the rat heart.

The level of myosin heavy chain (MHC) alpha mRNA and of MHC-beta mRNA was quantitated in the rat heart using a specific cDNA probe. In hypothyroid and diabetic hearts MHC-beta mRNA predominates, whereas in normal hearts MHC-alpha mRNA represents 80% of all MHC mRNA. Administration of 0.2 mg T3/100 g body wt. to hypothyroid rats led to an increase in MHC-alpha mRNA beginning at 3 h after injection and continued to rise until at 24 h control level of MHC-alpha mRNA were reached. In contrast, after administration of 2 units regular insulin to diabetic rats, MHC-alpha mRNA levels showed a small but significant increase already 30 min after insulin administration reaching a peak at 3 h and returning to diabetic values 5 h after insulin. The T3 response of other cardiac mRNAs was quantitated using in vitro translation, separation of 35S methionine labeled translational products and their quantitation by digital matrix photometry. An mRNA (spot 72b) coding for a translational product with a Mr 81,000 and pI of 5.4 showed a 3-fold increase in its level 1 h after T3 administration. In view of the rapid response of spot 72b and the early response of MHC-alpha mRNA to insulin, it is currently unclear if the T3 response of MHC-alpha mRNA represents a primary effect of T3.

Animals↗
PubMedDate not supplied

Renal morphology and function and urine electrolytes in experimental acute renal failure produced by cyclosporine and ischemia.

It has been difficult to produce a good animal model for cyclosporine nephrotoxicity. It has been suggested that by following 20 minutes of renal ischemia with four daily doses of cyclosporine 60 mg/kg intraperitoneally, one can create a model of reproducible renal failure. We observed excessive mortality (65%), due in part to cyclosporine's CNS effects, with these combined insults in the Munich Wistar rat. In contrast, cyclosporine alone in this dosage produced only 17% mortality and resulted in a similar degree of renal failure. Pair-fed and pair-watered vehicle and saline controls were used. The morphologic changes brought about by the castor oil vehicle of the parenteral cyclosporine solution were qualitatively similar to those brought about by cyclosporine by light microscopy, although the severity of the changes was considerably less in the vehicle-treated groups. However, by electron microscopy, pale lipid vacuoles were seen only in the cyclosporine-treated groups, whereas dense alterations in lysosomes and dilated endoplasmic reticulum also were seen in other groups. Urine sodium determined by flame photometry and urine chloride determined by Saltex reagent strips tended to be high in the initiation phase of cyclosporine-induced acute renal failure and low in the maintenance phase. In animals that developed acute renal failure following the combination of ischemia and cyclosporine, the initial urine sodium and chloride were significantly correlated with the eventual degree of renal failure. The use of Saltex urine chloride sticks in clinical urine samples showed that the readings correlated well with urine sodium and chloride determined by conventional methods, suggesting that these strips may be useful in making a quick diagnosis in the setting of acute renal failure.

Acute Kidney Injury↗
PubMed1979

Calcium measurements in serum and plasma--total and ionized.

This article will review the methods currently employed for measuring the concentrations of total and ionized calcium in serum or plasma. As far as total calcium is concerned, various techniques such as atomic absorption spectrometry, spectrophotometry, fluorometry, complexometric titration, and flame photometry will be described and compared. Particular emphasis will be given to the accuracy and precision of each technique. Possible sources of error and interfering agents will be identified and the various procedures for the taking and handling of blood samples evaluated. Inter-laboratory variation in the measurement of calcium will be studied. An assessment will be made of a new reference method for measuring total calcium in serum using isotope-dilution mass spectrometry. The usefulness of the total calcium measurement in clinical medicine will be briefly discussed. Within the last decade the refinement of spectrophotometric techniques and the improvements in ion-selective electrode technology have revolutionized the measurement of ionized calcium in serum, such that it may now be possible to replace total calcium measurements with ionized calcium measurements on a routine basis. The various techniques currently in use for measuring ionized calcium will be described and evaluated. Particular attention will be paid to the preparation of standards, the procedures for taking blood samples, and the handling of the samples prior to and during measurement. An assessment of the relative value of measuring total and ionized calcium will be presented.

Calcium↗