The kinetics of photopolymerization, in presence of riboflavin or riboflavin-5'-phosphate (FMN), have been studied either spectrophotometrically, at 283 nm, or chemically, by titrating unreacted double bonds with permanganate. The two sets of data are in good agreement and suggest that at least 8 h of light exposure are needed to ensure 95 % conversion of monomers into polymer. Up to now, it was generally accepted that photopolymerization should proceed for 1 h. It is not necessary to prolong light exposure for longer than 8 h, because even a 24 h illumination period only improves the conversion from 95 % to 96 %. Although conditions are described which ensure at least 95 % conversion of both riboflavin-and persulfate-catalyzed gels, their visco-elastic properties are widely different, suggesting that the final structure of these two matrices must vary considerably as a function of the catalyst used.

A precise and reproducible method for assessment of glycated hemoglobin in human adult red blood cells is reported, based on capillary isoelectric focusing (IEF). In order to obtain baseline resolution between adult hemoglobin (Hb A) and its glycated form (Hb A1c), two species which differ by minute delta pI values, < 0.03 pH units, the following procedure was adopted: the focusing mixture consisted of 5% Ampholine, pH 6-8, 0.5% Pharmalyte, pH 3-10, 3% short-chain liquid polyacrylamide and an equimolar mixture of two "separators", 0.33 M beta-alanine and 0.33 M 6-aminocaproic acid. The last two compounds flatten the pH gradient in the pI region of the two Hbs, thus allowing full separation. Additionally, the Hb samples, instead of being pulse-loaded, are uniformly distributed in the background electrolyte. A longer capillary life-time is obtained if all nonbuffering ions are eliminated; thus, as catholyte, 50 mM Lys (pH 9.7) is utilized and as anolyte 50 mM acetic acid (pH 3.5) is adopted. The percentages of Hb A1c, as obtained by capillary IEF, are in good agreement (+/- 6%) with data obtained by one of the standard zone electrophoretic methods in clinical chemistry, i.e., the Helena REP Glyco gel system.

As an alternative to the recently proposed screening for beta-thalassemias by isoelectric focusing (IEF) of denatured globin chains in urea-detergent gels [6], IEF of intact hemoglobins, obtained from umbilical cord blood in neonatal units, is reported here. For maximum separation, IEF should be performed in nonlinear pH gradients, in gels containing 0.2 M beta-alanine and 0.2 M 6-amino caproic acid, which flatten the pH gradient around pH 7, thus increasing the resolution between HbA and HbFac. The method can unambiguously detect homozygous and heterozygous beta-thalassemic conditions. A bimodal distribution of HbA at birth has been found: In heterozygous patients, HbA values of 9.02% (range 6.8-9.98%) have been found while in normal newborns HbA levels of 20.34% (range 11.02-30.6%) have been demonstrated.

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A new method for antenatal diagnosis of thalassemias is reported based on the analysis of the major Hb components of fetal cord blood, sampled at week 18 of pregnancy under ultrasonic guidance, by isoelectric focusing in immobilized pH gradients (IPG). In an IPG gel encompassing a pH 6.7-7.6 span, HbA and HbFac are separated by a distance nine times greater than in a conventional carrier ampholyte pH 6-8 gel and three times greater than in an ampholine gel with separators (an equimolar mixture of beta-alanine and 6-amino caproic acid). Band evenness (in terms of uniform protein concentration within a zone) and straightness (in terms of parallel alignment of the bands to the electrodes), because of insensitivity of IPG gels to salt distortions, allows for accurate and reproducible quantitation of HbF, -A, and -Fac levels. The possibility of greatly overloading IPG matrices in total Hbs increases the sensitivity of the technique to the detection of only 0.5% HbA in the total Hb mixture, the lower limit of conventional IEF being only 2.5% HbA. Of 15 fetuses from couples at risk analyzed in the region of Ozieri, three were found to be homozygous beta-thalassemic, eight heterozygous, and four normal with no false-positives or -negatives.