High-molecular intestinal alkaline phosphatase by agarose gel electrophoresis

The presence of high-molecular intestinal ALP (HIALP) overlapping with bone ALP in the alpha(2)beta region has been demonstrated. In this study we evaluated a method of separating HIALP after its conversion into ALP(5) by the action of protease. Serum samples from patients were mixed with protease at a ratio of 5:1 and left at room temperature for more than 30 min. The protease-treated and nontreated samples were both subjected to agarose gel electrophoresis. Patients who showed a decrease in ALP(3) in the alpha2beta region and an increase in ALP(5) in the beta region were regarded as HIALP-positive. HIALP was observed in 26.7-33.1% of patients with liver diseases, collagen diseases, and diabetes mellitus. Renal disease was ABO blood group-dependent and showed high positive rates for blood groups B and O. The HIALP-positive rate was low (7.1-15.5%) in patients with cardiovascular diseases, malignant tumors, and other disorders. ALP(5) was also observed in 98.4% of HIALP-positive patients with liver diseases. In patients with collagen diseases or diabetes mellitus, the positive rate of ALP(5) was 40.4-66.7%. In conclusion, this method, in which HIALP is converted into ALP(5) by protease pretreatment and is separated from bone ALP, allows HIALP to be identified while other fractions remain unaffected.     

Hyperlipoproteinemia phenotype determination by agarose gel electrophoresis updated

Hyperlipoproteinemia phenotyping has been re-evaluated by an improved agarose gel electrophoretic test. Eight varieties of serum lipoprotein electrophoretic patterns are demonstrated, two from clinically normal normolipidemic subjects and six from patients with hyperlipoproteinemia that include the phenotypes 1, 2a, 2b, 3, 4, and 5. The sharp delineation of the lipoprotein patterns achieved by this technique facilitates the differentiation and identifcation of the lipoprotein phenotypes. Furthermore, the lipoprotein patterns can be visually evaluated, correlated with their corresponding serum lipid values, and associated with the clinical and hereditary features of the hyperlipoproteinemia phenotypes. This test system can also be used in clinical trials and epidemiological studies.     

Determination of glycosylated hemoglobin by affinity electrophoresis on agarose gel

We describe a method for electrophoretic determination of glycosylated hemoglobin (GHb) on agarose gel membrane. Clear separation of GHb from non-GHb is achieved after 30 min electrophoresis at 60 V using a sodium citrate buffer solution (34 mmol/l, pH 6.5) containing 0.6 g/l dextran sulfate. The results obtained by this method correlate well with those by agar gel electrophoresis (r = 0.98) and column chromatography (r = 0.96). However, unlike column chromatographic methods, GHb values obtained on agarose gel are not affected by fluctuations in ambient temperature (18-28 degrees C), changes in pH (6.2-6.6) and ionic strength of buffer solution (26-40 mmol/l), or variant hemoglobin S and C. Also, electrophoresis on agarose gel membrane eliminates the lengthy preparative steps required for cellulose acetate electrophoresis. We conclude that agarose gel electrophoresis is a simple method for quantitative determination of GHb.     

Alkaline phosphatase isoenzymes resolved by electrophoresis on lectin-containing agarose gel

With this electrophoretic method the liver, biliary, and bone isoenzymes of alkaline phosphatase are clearly separated on agarose gels. Wheat-germ lectin, incorporated in the gel matrix, binds the bone isoenzyme selectively, forming a precipitate near the origin. Neither liver nor biliary isoenzyme is affected. Activity staining with an indigogenic dye substrate reveals the liver isoenzyme migrating nearest the anode, followed by the biliary and bone isoenzymes. Results are generally similar to those of electrophoresis on cellulose acetate. However, the lectin-agarose gels better resolve the liver and bone isoenzymes, and heat treatment of samples is not required before electrophoresis.     

Quantitative determination of high-density lipoprotein cholesterol by agarose gel electrophoresis.

We have developed a procedure for the determination of high-density lipoprotein cholesterol by agarose gel electrophoresis. Only 2 micro L of sample was applied to the gel. After electrophoresis at 90 V for 35 min, an enzymatic cholesterol reagent was applied. After a 30-min incubation, the high-density lipoprotein cholesterol was quantified by densitometry. Precision for this measurment approaches that reported for the heparin-manganese/Abell-Kendall method (Clin. Chem, 25: 596--609, 1979). We evaluated accuracy by comparing high-density lipoprotein cholesterol concentration measured by electrophoresis to that determined in the Framingham Heart Study procedure (J. Biol. Chem. 195: 357, 1952). The resulting correlation was excellent. By the paired Student's t-test, there was no significant difference between the two methods. The proposed method gives a linear standard curve when the concentration of total cholesterol is between 1.0 and 3.5 g/L. By accurate quantitation of high-density lipoprotein cholesterol, agarose gel electrophoresis can aid in assessment of coronary heart disease risk for a large segment of the population.     

Automated microdensitometry and quantification of lipoproteins by agarose gel electrophoresis.

A major obstacle in the application of quantitative microelectrophoresis has been tedious manipulations and calculations. To overcome these difficulties, we have developed an automatic system for the microdensitometry and calculations as part of a quantitative agarose gel electrophoresis facility. Results are internally standardized by serum cholesterol and/or triglyceride measurements. The hardware consists of a densitometer, an analog to digital converter, a cathode ray tube terminal, a teleprinter, and a small computer. A program in 4K words allows sample coding, electrophoretic scan display, indexing, and systematic identification of each peak. Data are acquired from scans of electrophoretic patterns of serum alone or in combination with the 1.006 gm/ml VLDL top and/or bottom preparative lipoprotein fractions. As many as 30 scans can be stored in 4K words of memory and then sent via high-speed telephone line to a larger computer for remote processing. The analysis corrects for baseline drifts and pre-beta asymmetry and will properly identify and quantify the amount of VLDL, LDL, and HDL with corrections for "sinking pre-beta" and "floating beta" in LDL and VLDL, respectively. Results are given in milligrams per 100 milliliter as well as percentile rank and standard deviation score ranking of each lipoprotein class as compared to an appropriate normal reference population. The latter data are in a form more meaningful to the physician and patient and provide a quantitative dimension to lipoprotein phenotyping.     

Two-dimensional gel electrophoresis, isoelectric focusing and agarose gel electrophoresis in the diagnosis of multiple sclerosis

Two dimensional gel electrophoresis (2-DE), isoelectric focusing (IEF) and agarose gel electrophoresis (AGE) were used to examine cerebrospinal fluid (CSF) and sera from 22 patients with confirmed multiple sclerosis, 11 patients with probable multiple sclerosis and 20 control patients with non-inflammatory neurological diseases of the central nervous system (CNS). All of the 22 patients with confirmed multiple sclerosis showed abnormal patterns of oligoclonal IgG in all three methods. In the CSF from patients with probable multiple sclerosis, oligoclonal IgG was detectable in 18 percent with AGE, in 72 percent with IEF and 90 percent with 2-DE. No oligoclonal IgG was observed in subjects with non-inflammatory neurological diseases. Many artefacts in IEF, which lead to misinterpretation, are eliminated in the 2-DE system. Based on our observations and this study in particular, it is evident that some patients have IgG changes which can be detected only by 2-DE. The application of research-oriented 2-DE for routine clinical purposes is still limited by its cost and technical complexity.