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The gas-liquid chromatographic measurement of serum creatinine is described in the present study. A preliminary concentration on cation exchange resin is used. The different steps of the operating procedure are analysed. A statistical comparison is undertaken between the results obtained with gas-liquid chromatography and kinetic colorimetry method using Jaffé assay.
We describe here a field method we developed for colorimetry of erythrocytic acetylcholinesterase (EC 3.1.1.7) in capillary blood samples. Three stable, premixed assay reagents and de-ionized water (but no centrifuge or balance) are required. This method, adapted for a microplate format, is essentially that of Ellman et al. (Biochem Pharmacol 1961;7:88-95) as modified by George and Abernethy (Clin Chem 1983;29:365-8). Assays were quantified and corrected for hematocrit by using a battery-powered colorimeter with a silicon carbide (blue) light-emitting-diode source. Advantages over existing field methods include better portability, ruggedness, greater precision, and lower cost per sample.
In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were determined in 33 subjects with breast cancer and compared to those of a group of healthy subjects previously described. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Mean plasma values of vitamin A, carotene and HDL-cholesterol were lower (P less than 0.01) than in the control group, the same applies to the RBP and PA mean levels (P less than 0.05). On the contrary, the mean value of total cholesterol was higher (P less than 0.01) in the patients than in the control group. Vitamin A plasma levels were significantly related to RBP and PA. No significant statistical correlation was found between clinical stage and vitamin A plasma levels.
Plasma vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were examined in healthy adult females. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Vitamin A and carotene mean values resulted as 43.0 +/- 8.2 micrograms/100 ml and 231.9 +/- 69.0 micrograms/100 ml, respectively. RBP and PA values averaged as 4.2 +/- 1.1 mg/100 ml and 29.4 +/- 6.1 mg/100 ml, respectively; whereas HDL-and total cholesterol were 179 +/- 16 mg/100 ml and 57 +/- 8 mg/100 ml. Vitamin A plasma levels were shown to be significantly related (P less than 0.01) to RBP and PA, but not to the other parameters examined (carotene, HDL-and total cholesterol).
Results of five non-specific reactions (ceruloplasmin assay, Ehrlich's reagent colorimetry, heat-resisting reaction of serum and determination of indole and neutral sulphide in urea) for the diagnosis of gastric carcinoma were calculated by the use of multiple-factor analysis. The positive rates in 284 cases of gastric carcinoma (including 8 early lesions), 306 healthy donors and 1018 cases of benign gastric diseases were 78.2% (62.5% for the early gastric cancers), 0.7% and 15.7%, respectively. The results showed that these tests are valuable for the screening in a high incidence area of gastric cancer or as a supplementary diagnostic means of gastric carcinoma in clinical practice.
I describe a new fluorometric method for determination of mandelic and phenylglyoxylic acids in urine, the fluorometry being preceded by extraction into ether and thin-layer chromatography. The chromatographically separated acids were quantitated after conversion to stable highly fluorescent derivatives by treatment with concentrated sulfuric acid. This method proves to be more precise, accurate, and reproducible than the existing colorimetric method. The limit of detection is 2 microgram of either acid per milliliter of urine with a CV of less than 15%. The standard curve for either acid is essentially linear from 2 to 100 microgram/mL of urine, with a correlation coefficient (r) of 0.97. No satisfactory correlation was obtained between the concentrations of either acid metabolite as measured in rat urine by fluorometry and colorimetry. The present method is considered suitable for routine biological monitoring of persons exposed to both high and low concentrations of styrene.
An accurate, reproducible method for less than or equal to 1 ppm iodine in foods is required for nutritional labeling. In order to ascertain the current status of iodine analysis in foods, 7 samples, representing different food classes, were analyzed by 8 laboratories. Six laboratories used their modifications of the Ce-As-I catalytic method preceded by alkaline dry ashing. Two laboratories used neutron activation analysis (NAA), with differing radiochemical separations. The study showed wide discrepancy in analytical results. Mean relative standard deviation for all laboratories was 77.9% between laboratories; 19.1% within-laboratories. Laboratories using NAA had only slightly better precision than did laboratories using the chemical method. The lowest level reported on the entire group of samples ranged among laboratories from 0.0089 to 0.65 ppm. Figures reported by a laboratory are, in general, consistently high or consistently low. The only differences in methodology which may possibly correlate with level of iodine obtained are the use of NAA technique and use of manual, rather than automated, colorimetry.
A simple and accurate method was developed for routine determination of fluoride in foods. Hydrogen fluoride is diffused 20 hr at 50 degrees C from fresh or freeze-dried samples (0.1 g dry wt) in polystyrene petri dishes containing 2 mL 40% HCIO4 and 0.3 g Ag2SO4, and is absorbed on the lids, previously spotted with 0.1 mL 0.5M NaOH. The absorbent layer is dissolved in 2 mL buffer solution, and the fluoride is measured potentiometrically. The method was verified by analysis of NBS Standard Reference Materials; recovery from 28 spiked infant foods (average = 99%, range = 75-135%); and comparison of results with colorimetry results for the same diffusates, after modification to handle 1 g samples. Relative standard deviations varied from 4 to 20% day to day. Detection limits were below 0.05 microgram/g dry weight.
A radioimmunoassay involving an 125I ligand has been developed and applied to the measurement of urinary metanephrine. To validate the clinical usefulness of this assay, we compared measurement of metanephrine by radioimmunoassay and of total urinary metanephrines by the Pisano colorimetric method. The radioimmunoassay is specific for metanephrine, whereas the colorimetric method measures both metanephrine and normetanephrine. We used both methods to determine urinary metanephrine or total metanephrines in subjects with essential hypertension, pheochromocytoma, the syndrome of multiple endocrine adenomatosis type 2, and normotensive volunteers. The mean and upper limit of normal (3 SD) for metanephrine by radioimmunoassay in our normotensive volunteers was 94.2 microgram/24 h and 229 microgram/24 h, respectively, which compares well with reported values of 87.6 microgram/24 h and 319 microgram/24 h by non-radioimmunoassay methods. Both radioimmunoassay and colorimetry accurately identified five patients with known pheochromocytoma. Good correlation (r = 0.993) was demonstrated between the two assays in a comparison of patients with essential hypertension and pheochromocytoma. We conclude that the radioimmunoassay is at least equivalent to the colorimetric methods in distinguishing pathological and normal catecholamine secretion, and is faster, more precise, and 1000-fold more sensitive.
We examined the effect of fetal hemoglobin and labile glycosylated hemoglobin on a number of diverse methods used to measure glycosylated hemoglobin. Samples were supplemented with various amounts of cord blood to give proportions of fetal hemoglobin ranging from 1 to 20% of total hemoglobin concentration. Procedures in which the separation of hemoglobin A1 from the major hemoglobin A fraction is based on differences in ionic properties (cation-exchange chromatography and electrophoresis) are subject to interference by fetal hemoglobin, whereas procedures that base the quantitation on other properties (colorimetry and affinity column chromatography) are not. The same procedures that are affected by the presence of fetal hemoglobin are also subject to interference by labile glycosylated hemoglobin. We conclude that the affinity chromatographic and colorimetric methods may give a more nearly accurate determination of glycosylated hemoglobin.
Microdeterminations for low molecular compounds highly contained in human seminal plasma, i. e., dehydroepiandrosterone sulfate (DHEA-S), spermine (Sp) and citric acid (CA) have been established. Experimental studies were carried out on application of these methods to identification of semen from cadaveric vaginae more than a few days after death. DHEA-S was measured by radioimmunoassay using anti-DHEA-S-17-hydrazone conjugated bovine serum albumin rabbit antiserum. Sp and CA were determined spectrophotometrically with monoamine oxidase and with citrate lyase, oxalacetate decarboxylase and pyruvate oxidase, respectively. DHEA-S and its cross-reacting materials were contained little in saliva, moderately in semen and vaginal contents and largely in serum and urine. By the above method Sp was not detectable in other body fluids except semen. CA was contained not only largely in semen but also a little in other body fluids. DHEA-S and Sp in the seminal fluid allowed to stand at room temperature were relatively stable during a month, whereas CA became undetectable in 10 days, all of which being persistent in seminal stain during at least one month. From these results the colorimetry of Sp among the three methods above mentioned was found to be most reliable for detecting seminal fluid from vaginal contents of a putrefied cadaver.
The determination of glycosylated hemoglobin (GHb) has been found useful in the medium term assessment of diabetic control. Levels of GHb have been shown to be influenced by hematological abnormalities and methodology amongst other factors. Hemoglobinopathies are common in Nigerians and other people of African descent. No work, to our knowledge, has been done in Nigeria to study the effect of hemoglobinopathies on the levels of GHb in Nigerians or Africans. In this study, GHb levels in non-diabetic Nigerians (with hemoglobin genotypes AA, AS, and SS) were determined by short column chromatography and thiobarbituric acid colorimetry. Levels of GHb obtained by the microchromatographic method were significantly different from one another in the three subject groups. The group mean GHb level was highest in subjects with sickle cell anaemia and lowest in subjects with sickle cell trait. However, using the colorimetric method, the mean GHb level of the normal subjects with hemoglobin genotype AA was not significantly different from that of subjects with hemoglobin genotype AS or SS. Results of GHb determinations by microchromatography (for which several commercial kits are available) in subjects with sickle cell anemia or trait must be interpreted with caution. The colorimetric method, though more tedious, gives more reliable results and should be the method of choice in subjects with concurrent diabetes and sickle cell anemia or trait.
Dry, thin films containing all necessary reagents for clinical analysis by colorimetry have been designed. Reagents in a matrix of hydrophilic polymer are coated on top of a transparent plastic base. A white isotropically porous polymer spreading layer, 80% void volume, is coated over the reagent layer(s). In the analysis, a drop (typically 10 microliter) of undiluted serum or other fluid is touched to the spreading layer. The fluid spreads rapidly and uniformly through the pore structure, filling a void volume corresponding to the drop volume. Water and low-molecular-weight components diffuse from the spreading layer into the reagent layer(s), initiating the reaction sequence. The spreading layer acts also as a white optical diffuser for reflection densitometry. Optical reflection density is linearized through use of the function developed by Williams and Clapper [J. Opt. Soc. Am. 43, 595 (1953)] to convert reflection to transmission density. A wide variety of chemical assays are compatible with this format. As an example, for the glucose film we found coefficients of variation of 1.5% in predicting glucose concentrations in control sera during 20 days. Results for glucose concentrations in several hundred patients' sera by the present method were very cose to those obtained with the Center for Disease Control's hexokinase reference method.
For this direct colorimetry of urinary oxalate, commercially available oxalate oxidase (EC 1.2.3.4) is used. The urine is first diluted, to diminish the effect of interfering substances. Analytical recovery of oxalate from urines with five different oxalate concentrations (0.4 to 2.0 mmol/L) ranged from 92 to 109% (mean 99%). The within-day and between-day precision (CV) of the method for a wide range of oxalate concentrations averaged better than 10%. There is good correlation (r = 0.977) between this enzymatic method (y) and the chemical method of Hodgkinson and Williams (x) [Clin Chim Acta 36: 127-132, 1972], the regression equation being y = 1.014x + 0.061. Urines with added ascorbate give falsely increased results. The proposed method is inexpensive and simple to perform.
We describe two fully enzymic methods, fluorometric and colorimetric, for determination of triglycerides (triacylglycerols) in serum. Samples are incubated with microbial lipase for 10 min, and the glycerol released from the triglycerides is oxidized by NAD+ in the presence of glycerol dehydrogenase. In the fluorometric method, the resulting NADH is in turn oxidized by resazurin as catalyzed by diaphorase to form resorufin, a highly fluorescent compound. In the colorimetric method, the NADH is oxidized by coupling with a tetrazolium salt/diaphorase system to form formazan, a highly colored compound. Calibration curves, constructed by plotting change in fluorescence or absorbance vs concentration of triglycerides, were linear up to 6 and 5 g of triglycerides per liter of serum for the fluorometric and colorimetric methods, respectively. The assays require only 5 and 15 microL of serum for fluorometry and colorimetry, respectively. The CV was 0.59% for the fluorometric method, 0.91% for the colorimetric procedure. The time for analysis for either method is less than 15 min. The results correlate well with those obtained by the Dow Diagnostic Kit method, a colorimetric method in which glycerol kinase and glycerol-1-phosphate dehydrogenase form NADH from ATP and NAD+ in the presence of glycerol and glycerol 1-phosphate.
We describe a liquid-chromatographic procedure for determining urinary p-aminobenzoic acid (PABA) in the exocrine pancreatic function test. The urine specimen, collected for 6 h after oral administration of N-benzoyl-L-tyrosyl-p-aminobenzoic acid, is hydrolyzed in 4 mol/L NaOH containing m-hydroxybenzoic acid as internal standard, to free PABA metabolites, then diluted and injected into the chromatograph. PABA and the internal standard are eluted from a reversed-phase C18 column with phosphate buffer (0.2 mol/L, pH 3.5)/acetonitrile, 87.5/12.5 (by vol), and detected with an electrochemical detector at +1100 mV. Urinary PABA concentration as measured by this method agreed well with that by colorimetry involving p-dimethylaminocinnamaldehyde. The present procedure is specific for PABA, precise, and fairly rapid. We hope that the present method will minimize instances of interference from drugs, which cannot be avoided in the colorimetric method.
Iron in serum and urine specimens containing deferoxamine (a chelator used in treating acute iron intoxication) can be measured by the DuPont aca discrete-analyzer method for iron if 0.5 mL of a 100 mL/L solution of thioglycolic acid (mercaptoacetic acid) is added to the IRN-1 and IRN-2 test packs before their use. This reagent releases the unreactive chelated iron, allowing reliable colorimetry. Because about 50% of hemoglobin iron is measured by the modified method, hemolysed samples should not be used (or results should be corrected for hemoglobin iron). The modification allows emergency determination to total serum iron and assessment of urinary iron during chelation therapy, as well as measurement of plasma iron in samples treated with sodium citrate or oxalate plus fluoride (the unmodified aca iron method gives falsely low iron values in the presence of these chelators). About 60% of iron present as the dextran complex (Imferon) is measured by the modified aca method, as compared with <5% by the unmodified aca technique.
The method of enzyme activity related in enzyme immunoassay was described. The detection methods were colorimetric, fluorometric and chemi- or bioluminometric assay for enzyme, and the sensitivity of three detection methods were compared. The order of detection limit was chemi- or bioluminometry > fluorometry > colorimetry. But it is important that the selection of detection method of enzyme will make fit on your desirable sensitivity of analysis.