The biochemistry, laboratory determination, and clinical or laboratory applications of very small quantities of hemoglobin are reviewed. No single assay method is suitable for the whole range of low concentrations, and for the forseeable future, as uniform standards are unlikely to be generally accepted, each laboratory must establish its own. Nevertheless, the development of an assay for very small amount of hemoglobin will be an important advance in many fields. There was a time when the chromogenic methods using benzidine and some part of its derivatives were used widely, because of their high sensitivity. However, since it was found that these chromogens were carcinogenic, many investigators have studied and reported alternative safety and sensitive methods. Here, based on these methods, we recommend the best available means of assessing the control of hemolytic anemia, screening for colorectal cancer, and monitoring blood storage.
Excretion and heterogeneity due to varied carbohydrate structures of both active and latent types of human urinary kallikrein (HUK) in normal subjects and some patients were investigated. Both active and latent types of HUK excretions were decreased in essential hypertension, chronic glomerulonephritis, chronic renal failure andnephrotic syndrome compared with normal subjects. In contrast, both types of HUK excretions were markedly increased in Bartter's syndrome. Analysis of sugar moieties of two types of HUKs in normal subjects and patients urine was carried out by the methods of seriallectin affinity chromatographies and crossed affino-immunoelectrophoresis. The analytical results derived from two different methods were almost agreed, especially in two diseases, i. e. essential hypertension and Bartter's syndrome, the alterations of chromatographic and electrophoretic patterns were observed and are supposed to correspond to glycosylation changes in each HUK.
Serum concentrations of α-carotene, β-carotene, lycopene, and retionl-binding protein (RBP) in healthy adults were determined by HPLC or immunodiffusion to be the following: 6.7±4.8μg/dl, 22.8±16.2μg/dl, 26.3±17.9μg/dl, and 5.0±0.9μg/dl, respectively, for males (n=65); and 7.9±4.0μg/dl, 43.8±21.4μg/dl, 28.8±15.6μg/dl, and 3.8±0.7μg/dl, respectively, for females (n=81). Serum β-carotene concentrations were significantly higher in females than in males (p<0.001), while serum RBP values were lower in females than in males (p<0.001). The differences between the sexes in β-carotene and RBP concentrations were apparent in subjects over 15-years of age. Moreover, concentrations of β-carotene and α-carotene were higher with increasing age in females, but not in males. On the other hand, serum RBP values in males were higher with increasing age. Serum lycopene concentration was at its peak between the ages of 20 and 30 for both males and females. In addition, serum concentrations of α-carotene, β-carotene, lycopene, and RBP in outpatient subjects aged from one month to 19 years changed according to advancing age in both males and females. No differences in sexes for these mean values were found for subjects less than 14 years of age; they appeared later along with the secondary sex characteristics.
In order to detect assay abnormality or to analyze patients' data, assayed data are transformed using biased log transformation log (x+A), and the transformed data are observed on the two variable correlogram. One half of the normal range of each test is used as the bias A. The ratio of highly correlated tests are commonly used to detect abnormal assay results or specified pathological condition. By the observation of the correlogrom, it was found that some of the combination of two tests are not proportional each other, but showed approximately powered function relationship. Upper and lower limits of data distribution can be drawn on the correlogram and the data out of limits are analyzed. Many of them represents special pathological state of the patients, but not assay abnormality. The data distribution model of log-normal distribution with assay error is calculated with numerical integral, and it is proved that the distribution is well simulated with biased log-normal distribution. The value of the bias (A) is proportional ro square of the assay error.
A high-performance liquid chromatographic method was established for detecting coproporphyrin I and In isomers. Coproporphyrin I and III isomers were separated on a reversed-phase column with isocratic elution and measured by fluorometry. The recovery rate of each coproporphyrin isomer was nearly 100% and the minimum detection limit was less than 1μg/l in urine. The normal ranges (95% limit) were estimated to be 22.2-57.4μg/g creatinine for coproporphyrin I, 22.1-109.1μg/g creatinine for coproporphyrin III, and 0.71-3.73 for coproporphyrin III/coproporphyrin I ratio.
Recently, desmosine (D) and isodesmosine (ID) in elastin hydrolysates have been analyzed by high-performance liquid chromatography (HPLC). However, it is difficult to resolve ID and ID by these procedures and the regular HPLC procedures themselves are time-consuming. For these reasons, a rapid and simple determination of D anb ID dy HPLC was developed. D and ID were separated on an HPLC column (Nucleosil C-18) with a mobile phase of 0.1 M CH3SO3H(pH 2.0)/CH3CN=90/10 containing 6 mM heptane sulfonate or 90/5 containing 2 mM heptane sulfonate. We applied the method for the analysis of D and ID contained in the elatstin hydrolysates of bovine ligamentum nuchae, smooth muscle culture medium, and rat aorta. Results of the analysis of the ligamentum nuchae and the medium agreed with those obtained by automatic amino acid analysis. The method has advantages over other types of analysis for D and ID. It is accurate, reproducible, and simple and allows rapid analysis. Furthermore, its sensitivity is such that elastin from small amount of tissue that containslow concentrations of elastin can be quantitated. The determination of D and ID in tissues or cell culture medium containing elastin may assume the same significance as the determination of collagen based on its hydroxyproline content.
The authors deviced the assay method for the ratio (Fe2+/Fe3+) of iron in iron binding human serum protein with Nitroso-PSAP and ascorbic acid. The method consists of (1) elimination of reducing agent (ascorbic acid etc.) from serum protein by the centrifugation with Centriflo membrane cone,(2) liberation of iron (Fe2+, Fe3+) from serum protein by addition of deproteinizing solution cone,(3) determination of Fe2+ with Nitroso-PSAP, and (4) successive determination of Fe2+ formed from Fe3+ by addition of ascorbic acid. The ratio (Fe2+/Fe3+) of iron bound with protein in sera of 14 healthy subjects at 6 pm was 1.28±0.92 (mean±SD). The ratio in each serum was not changed when the serum samples were stored at 2°C or-13°C for at least 6 days.