Demonstration of 2-hydroxybenzoylglycine as a drug binding inhibitor in newborn infants.

Newborn infants have drug binding defects that share similarities to those of uremic subjects. Since 2-hydroxybenzoylglycine has been chemically defined to be a major drug binding inhibitor in uremia, a search for the presence of a similar compound in the sera of newborn infants was made. An organic substance that has the characteristics of 2-hydroxybenzoylglycine as supported by the retardation factor values on thin-layer chromatograms, retention times of high performance liquid chromatograms, fluorescence emission spectra, and mass spectrum has been demonstrated to be present in the majority of the neonatal sera studied. A strong positive correlation between the levels of the binding inhibitor and the extent of binding defects for nafcillin has been observed. The substance could effectively reduce the total bilirubin concentration when added to the cord sera specimens. It is concluded that 2-hydroxybenzoylglycine plays an important role in drug binding defects observed in the newborn, and the inhibitor may also play a part in the precipitation of bilirubin-induced neurotoxicity in neonates when the substance is abnormally elevated.

Isolation and chemical characterization of 2-hydroxybenzoylglycine as a drug binding inhibitor in uremia.

An organic compound that inhibits drug binding in uremia has been isolated from the sera of chronic renal failure patients, and its chemical structure has been determined. Addition of the compound to normal human sera in vitro resulted in drug binding defects similar to those seen in uremia. The purification of this substance was accomplished by n-butyl chloride extraction of acidified (pH 3.0) uremic sera followed by column chromatography, thin-layer chromatography, and paper electrophoresis. From analytical studies including ultraviolet and fluorescence spectroscopy, gas chromatography, chemical ionization and electron impact mass spectrometry, and proton nuclear magnetic resonance spectroscopy, the chemical structure of the uremic binding inhibitor was deduced to be 2-hydroxybenzoylglycine. This confirms the hypothesis that the drug binding defect in uremia is due to the accumulation of endogenous metabolic products rather than an intrinsic structural defect in albumin.

Correction of drug binding defects in uremia in vitro by anion exchange resin treatment.

Serum protein binding of weakly acidic drugs is impaired in uremia, but that of basic drugs tends to be normal. Treatment of uremic serum with anion exchange resin (Amberlite CG-400, acetate form) corrected binding defects for three acidic drugs (nafcillin, salicylate and sulfamethoxazole) but did not affect the binding of two basic drugs (trimethoprim and quinidine). Resin treatment of normal human serum did not alter the binding of these five drugs. Extraction of the acetate buffer eluate from resin exposed to uremic serum with n-butyl chloride at acidic pH (3.0) resulted in a fraction that could induce similar binding defects in normal human serum. The factor(s) responsible for binding defects in uremia appears to be lipid soluble, weakly acidic, and dialyzable. It is believed to be tightly bound to albumin at physiologic pH, but dissociates from it at acidic pH. These findings further support the previously proposed hypothesis that drug-binding defects in uremia are due to accumulation of certain endogenous metabolic product(s).

Partial purification and characterization of the drug-binding-defect inducer in uremia.

The chemical basis of drug-binding defects in uremia was investigated by studying the effects of extraction of uremic sera with an organic solvent (n-butyl chloride). Extraction of uremic sera at acidic pH (3.0) with n-Butyl chloride fully corrected the binding defects for three acidic drugs (nafcillin, sulfamethoxazole, and salicylate), whereas binding of two basic drugs (trimethoprim and quinidine) was unaffected by similar treatment. When added to normal human serum or purified human serum albumin, the organic solvent layer was capable of inducing binding defects similar to those seen in uremia. Further fractionation of the organic solvent layer with purified human serum albumin at physiologic pH (7.4), followed by reacidification and extraction of the acidified albumin layer with the same solvent, gave a homogeneous fraction on thin-layer chromatography. This homogeneous fraction could induce the binding defects observed in uremia when added to normal human sera. This factor(s) is apparently a dialyzable and weakly acidic compound. It is lipid-soluble and tightly bound to albumin at physiologic pH, but extractable at acidic pH. Its molecular weight is approximately 500 or less. These findings strongly support the hypothesis that the drug-binding defect in uremia is due to accumulation of endogenous metabolic products rather than to an intrinsic structural abnormality in serum albumin.

Rapid assay for determination of trimethoprim and sulfamethoxazole levels in serum by spectrofluorometry.

A rapid spectrofluorometric method for determining the levels of both trimethoprim and sulfamethoxazole from the same specimen of serum is described. The method involves stepwise extraction of the specimen first with chloroform at an alkaline pH (pH 9.0) for trimethoprim followed by n-butyl chloride at an acidic pH (pH 2.0) for sulfamethoxazole. To quantitate trimethoprim, the chloroform layer was subjected to fluorometry by exciting the specimen at 295 nm and measuring the relative intensity at 330 nm. To determine sulfamethoxazole levels, the n-butyl chloride layer was subjected to fluorometry by exciting the specimen at 285 nm and measuring the relative intensity at 330 nm. Relative intensities were linear (r greater than 0.99) over the concentration ranges of 0.5 to 40 microgram/ml for trimethoprim and 1 to 400 microgram/ml for sulfamethoxazole. Values obtained by this spectrofluorometric procedure were in excellent agreement with those obtained by a conventional fluorometric assay for trimethoprim and a colorimetric assay for sulfamethoxazole. Elevated levels of endogenous metabolic products and numerous other drugs, including a number of antimicrobial agents, did not interfere with the method. Although salicylates interfere with the determination of sulfamethoxazole, an appropriate correction can be made. This method can also be used to determine the drug levels in cerebrospinal fluid.

New rapid assay for nafcillin in serum by spectrofluorometry.

A new, rapid method for measuring serum levels of nafcillin by spectrofluorometry is described. The method involves extraction of 2 ml of acidified serum with n-butyl chloride, subjecting the organic solvent layer to excitation at 340 nm, and measuring the relative intensity of emission fluorescence at 380 nm. An excellent linear correlation exists between serum levels of nafcillin and the relative intensity in a drug concentration range of 0.25 to 150 mug/ml. The results obtained by this spectrofluorometric technique are in complete accord with those obtained by the conventional microbiological assay using Staphylococcus aureus ATCC 6538P. The method is not interfered with by elevated levels of endogenous metabolic products or the presence of other drugs, including a number of antimicrobial agents. The assay is interfered with, however, by the presence of salicylates, for which appropriate correction can easily be made. A salicylate assay employing a spectrofluorometric technique is also described.