Using the general concept of a dry multilayer analytical element, we can change chemical procedures and configurations to assay several blood components. In the assay of serum urea nitrogen, urease in the reagent layer catalyzes the hydrolysis of urea. A semipermeable membrane excludes aqueous base, but allows ammonia to diffuse to an underlying indicator layer. For the amylase determination, the enzyme hydrolyzes a dyed-starch substrate coated on top of the spreading layer; this produces small fragments, which diffuse to a registration layer. The increase of absorbance at 540 nm is correlated with amylase activity. Bilirubin complexes with a cationic polymer at the interface between the spreading and reagent layers. The direct reading at 460 nm allows determination of total bilirubin in the range 1 to 500 mg/liter. Tirglycerides are hydrolyzed in the spreading layer, and the resulting soluble glycerol readily diffuses into the reagent layer, where it is phosphorylated and subsequently oxidized by glycerophosphate oxidase to yield dihydroxyacetone phosphate and hydrogen peroxide. Peroxidase catalyzes production of a color commensurate with the hydrogen peroxide produced.
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.
Delta bilirubin is a strong, possibly covalent complex between bilirubin and an albumin-like protein, found in the serum of most jaundiced adults. The bili-albumin complex reacts directly diazo-positive. An examination of its azo pigments indicates that only one half of the tetrapyrrole is linked to the protein, presumably via an amide bond formed between a propionic acid side chain of bilirubin and an amine group (e.g., E-NH, of a lysine) in the albumin backbone; the other (distal) propionic acid chain is largely unesterified. Preliminary analyses of the peptides generated by chemical or enzymic cleavage of the delta fraction suggest a bilirubin bonding region within positions 128-297 of albumin and another involving positions 298-585. Thus, delta bilirubin may represent the first true human biliprotein. The implications of this biliprotein in both the fundamental and applied aspects of bile pigment research are discussed.
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