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The molecular defects of three different slow-migrating genetic variants of human serum albumin, albumins Kamloops (formerly RIH), Stirling and Amsterdam, previously characterized only by electrophoretic and dye-binding studies, are now reported. Two of them are proalbumin variants: sequential analysis of the purified whole proteins has established the mutation responsible for albumin Kamloops as -1Arg-->Gln, and for albumin Stirling as -2Arg-->His. A Glu-->Lys substitution in position 570 of the mature albumin molecule was determined in albumin Amsterdam by sequential analysis of two abnormal tryptic fragments. The three alloalbumins are caused by single-base changes all of which seem to represent hot-spots in the albumin gene. The possible functional consequences of the presence of a circulating alloalbumin are discussed.

An early case of bisalbuminaemia was reported in this journal in 1964, with the name Albumin Reading added later. Its use in electrophoretic comparisons led to some new variants being described as 'of the Reading type' on this basis alone. Protein sequencing and DNA studies have since found the single point mutation 313 Lys-->Asn common to this type, but the eponymous variant has not, until recently, been available for study. We now report on its characterisation using PCR analysis with allele-specific oligonucleotide primers, a method also applicable to studies of the population distribution of variants. We also draw attention to the need to link clinically-significant effects to individual variants of known structure.

In the circulation, non-esterified fatty acids are transported by albumin which also facilitates their removal from donor cells and uptake into receptor cells. We have studied whether genetic variations in the albumin molecule can affect its in vivo fatty acid-binding properties. The fatty acids bound to 25 structurally different variants and to their wildtype counterparts, isolated from heterozygous carriers, were determined gas chromatographically. The variants were proalbumins, albumins with single amino acid substitutions and glycosylated or truncated albumins. In eight cases the total amount bound to the variants was diminished (0.4-0.8-fold), and in seven cases the load was increased to 1.3 or more of normal. Twenty-one fatty acids were quantitated, and for 19 alloalbumins significant deviations from normal were found. Usually, changes in total and individual fatty acid binding were of the same type, but several exceptions to this rule was found. The glycosylated albumin Casebrook showed the largest changes, the total load and the amount of bound palmitate was 8.6 and 14 times, respectively, the normal. The most pronounced changes and the majority of cases of increased binding were caused by molecular changes in domain III. Mutations in domain I, II and the propeptide resulted in smaller effects, if any, and these were often reductions in binding.

Binding of laurate (n-dodecanoate) to genetic variants of albumin or its proprotein and to normal albumin isolated from the same heterozygous carriers was studied by a kinetic dialysis technique at physiological pH. The first stoichiometric association constant for binding to proalbumin Lille (Arg-2-->His) and albumin (Alb) Roma (Glu321-->Lys) was increased to 126% and 136% respectively compared with that for binding to normal albumin, whereas the constant for Alb Maku (Lys541-->Glu) was decreased to 80%. In contrast, normal laurate-binding properties were found for as many as nine other albumin variants with single amino acid substitutions. Because the net charges of all these mutants were different from that of normal albumin, the results suggest that the examples of modified laurate binding are not caused by long-range electrostatic effects. Rather, the three positions mentioned are located close to different binding sites for the fatty acid anion. The most pronounced effect was observed for the glycosylated Alb Casebrook, the binding constant of which was decreased to 20%. Binding to the glycosylated Alb Redhill was also decreased, but to a smaller extent (68%). These decreases in binding are caused by partial or total blocking of the high-affinity site by the oligosaccharides, by the negative charges of the oligosaccharides, and/or by conformational changes induced by these bulky moieties. Laurate binding to two chain-termination mutants (Alb Catania and Alb Venezia) was normal, indicating that the C-terminus of albumin is not important for binding. By using different preparations of normal albumin as controls in the binding experiments, it was also possible to compare the effect of various methods for isolation and defatting on laurate binding.

The study of human serum albumin variants is reviewed with reference to albumin Kashmir, a typical variant. Its published instances are listed and its position in this field of investigations is indicated.

The molecular basis for albumin Kashmir was studied using the polymerase chain reaction to amplify a DNA fragment containing codon 501 in exon 12 of the human albumin gene. Southern blots of the amplified DNA were hybridized to oligonucleotide probes specific either for the normal allele of albumin or for albumin Kashmir. The results provide strong evidence that codon 501 in albumin Kashmir is AAG (lysine) instead of GAG (glutamic acid), thus confirming the protein sequences reported. This approach was used to characterize a bisalbuminaemic individual as a carrier for albumin Kashmir. Similar strategies may be devised to study the molecular basis and to identify carriers of other alloalbumins.

Six tests recommended for use in the diagnosis of cystic fibrosis (CF) have been compared in the same subjects. The tests were carried out on 165 normal persons, 64 known cases of CF, their 67 parents and 18 sibs. The tests measured sodium in fingernails by activation, sodium in saliva and chloride in saliva and in thermal and pilocarpine-induced sweat by means of ion-specific electrodes, and chloride in pilocarpine-sweat by the standard titrimetric method. The tests ranged in complexity from simple screening methods to individual clinical procedures. None of the simpler tests matched the standard pilocarpine method in diagnostic efficiency, whether they were used singly or in combination.

Of 18 members of a Fiji Indian family investigated, eight of the 12 males and two of the six females had an electrophoretically slow-type bisalbuminemia (alloalbuminemia). The albumin was characterized by the hiterto unique ratio of the two bands (Al A 35%: variant 65%), and by dye-binding studies and electrophoretic mobility in different media. The data suggest that this is a new variant, which we propose to call albumin Vancouver (Al Va).

A case of bisalbuminaemia has been found in Luarca, Asturias. At least two relatives also bear the trait. The variant albumin Luarca (Al Lu, gene notation Al(Lu)), the first to be found on the mainland of Spain, is described in terms of its electrophoretic mobilities and dye-binding properties. It is heat-stable but sensitive to storage, freezing, and thawing and it is the first albumin to show this property.