Nonenzymic glycation of human immunoglobulins does not impair their immunoreactivity.

Diabetic patients have an increased proportion of their immunoglobulins nonenzymically glycated. To investigate the possibility that this may contribute to increased susceptibility to infection, we compared the immunoreactivity of glycated and nonglycated human immunoglobulins against rubella and hepatitis; streptococcal exoenzyme and infectious mononucleosis; human lymphocytotoxic antigens (HLA); and Varicella zoster in terms of antigen-antibody binding, cell agglutination, cytotoxicity, and complement-fixation properties, respectively. We found no evidence to support the supposition that glycated immunoglobulins are functionally impaired.

Capacity related characteristics of Glyco-Gel affinity chromatographic support.

We examined capacity related properties of "Glyco-Gel" (Pierce), a boronate agarose gel for separating and measuring glycated proteins by affinity chromatography. Our data indicate linear capacity to as much as 20 mg as applied hemoglobin or almost 10 mg as bound hemoglobin and 26 mg as applied serum proteins or a minimum of 2.5 mg as bound serum protein for each mL of gel. The capacity and affinity of the support for glycated proteins becomes optimum only after four regeneration cycles. The support matrix appears to have a small concentration of nonspecific binding sites equivalent to 0.09 to 0.18 mg as serum protein for each mL of gel. These sites do not bind hemoglobin. They lead to an overestimation of glycated protein that can cause large errors when the proportion of glycated protein is determined with small column loads. If near capacity loads are applied, the samples must be dialyzed or diluted to avoid decreased analytical recovery resulting from competitive and eluting properties of endogenous sugars.

Non-enzymic glycation of individual plasma proteins in normoglycemic and hyperglycemic patients.

Diabetic patients in poor glycemic control show increased glycation of total plasma proteins, but little is yet known about the relative extents to which the various individual proteins are glycated. Thus, we studied the non-enzymic glycation of several major plasma proteins and plasma protein fractions in normal and diabetic patients. In vivo glycation for most plasma proteins was very low in non-diabetic patients, only gamma globulin showing more than 5% glycation. In diabetic plasmas, glycation was much greater, immunoglobulins again showing the greatest proportion, followed in descending order by albumin, complement C3, fibrinogen, transferrin, haptoglobin, and alpha-1-antitrypsin. When plasma proteins were glycated in vitro, this order was IgG greater than complement C3 greater than albumin greater than transferrin greater than haptoglobin greater than alpha-1-antitrypsin. In general, proteins with the longest biological half-lives, such as IgG and albumin, showed the greatest in vivo glycation. On the other hand, proteins with high intrinsic glycability, such as complement C3, showed moderate glycation, despite a short half-life. Except for albumin, more basic proteins showed greater glycation than acidic proteins, but there was poor correlation between mole percent lysine and glycation. Evidently the relative extents of glycation of different plasma proteins are a complex function of integrated glucose concentrations over time and of the half-life and chemical characteristics of each protein.

Nonenzymatic glycation of immunoglobulins does not impair antigen-antibody binding.

We glycated immunoglobulins from commercial kits designed to measure human ferritin, thyrotropin, and transferrin, and compared the calibration curves for assays utilizing glycated antibodies with those of assays utilizing non-glycated antibodies. Glycation was verified by borate affinity chromatography and assay with thiobarbituric acid reagent. We found no evidence that antigen-antibody binding is impaired by nonenzymatic glycation of antibodies. Our results provide no evidence in support of the supposition that glycation may be a contributory factor in the decreased resistance of diabetics to infection.

Improved separation of fetal hemoglobin by mini-column chromatography.

We present chromatographic conditions for separating and measuring fetal hemoglobin on a 5-cm column of Sephadex CM-50. A distinct fetal band is evident, even when present at less than 1% of total hemoglobin. Precision (CV) of better than 5% is achievable.

A "column-batch" method for separating MB and LD1 in a single fraction.

In this "column-batch" method for separating the MB and BB isoenzymes of creatine kinase and the LD1 isoenzyme of lactate dehydrogenase, one can, alternatively, separate MB from BB or obtain a combined fraction containing MB, BB, and LD1. The principal advantage is that the resulting fractions are twofold as concentrated as was the applied sample. Thus, activity can be measured by conventional automated methods, with no need for the modifications to compensate for diluted fractions that are required by other ion-exchange methods. Another advantage is the total absence of interference by the MM isoenzyme. A strong anion exchanger (AG-MP1, Bio-Rad) is used in the acetate form at pH 6.3. There is no retention of MM; retained MB, BB, and LD1 are eluted with a solution of magnesium acetate. Results are compared with those obtained for subunit B and LD1 by immunoinhibition. Results with patients are considered consistent with myocardial infarction if MB exceeds 20 U/L and 3% of the total CK and LD1 exceeds 130 U/L or 28% of the total LD activity.

Stability of creatine kinase-3 in lyophilized materials.

We examined the stability of creatine kinase (EC 2.7.3.2) isoenzyme-3 (CK-3) in lyophilized bovine albumin matrices in the presence and absence of various sulfhydryl compounds and ADP. We initially purified CK-3 from human myocardium and skeletal muscle by the batch-chromatographic technique and by gradient elution column chromatography to specific activities of 293 and 93 kU/g, respectively. To assess stability, we subjected the lyophilized materials to storage studies at 4, 25, 37, 42, 56, and 65 degrees C and compared first-order rate constants for the decay of creatine kinase activity at 42 degrees C. Our most stable matrix contained, per liter, 2 mmol of ADP and 10 mmol of N-acetylcysteine, and had an extrapolated first-order half-life (Arrhenius plot) at -20 degrees C of approximately 60000 years.

In search of optimum conditions for the measurement of creatine kinase activity: a critical review of nineteen formulations.

We compare the reagent composition recommended by six different groups including three European societies for the determination of creatine kinase activity in serum using the coupled hexokinase/glucose-6-phosphate dehydrogenase (EC 2.7.1.1/1.1.1.49) reactions. Even though discrepancies exist between these methods, there are, nevertheless, major areas of consensus which permit a reasonable extrapolation of an approximate composition for optimum response. We then ascertain how reagents used in thirteen commercial kits differ from these approximated optimum conditions. Except for four companies, all the reagent compositions differ remarkably from the conditions recommended by the six groups.

Creatine kinase isoenzyme--antibody reactions in immuno-inhibition and immunonephelometry.

I have raised, in rabbits, antibodies against MM and BB isoenzymes of creatine kinase. The antibodies produced were homogeneous by Ouchterlony double immunodiffusion and did not cross react with their opposite antigens. Both antisera, however, appeared to be mixtures of antibodies, displaying different equivalences for activation, inactivation, and, possibly, precipitation. Inactivation studies indicated the presence of antibodies effective against dimer only and antibodies effective against monomer or dimer. Both antisera cross reacted with MB and displayed antibodies that appeared to block only half of the activity as well as antibodies that blocked all of the activity. The antisera produced were useful for measuring MB by both immuno-inhibition and immunonephelometry, but neither appears to be advantageous over current electrophoresis or ion-exchange methods. A comparison of decay of MB in patients between activity and mass measurements indicated that activity decay is about 12-fold faster than mass decay.

Creatine kinase: re-examination of optimum reaction conditions.

I have re-examined optimum reaction conditions for measurement of creatine kinase (EC 2.7.3.2). The optimum pH is 6.45, and 2,2-bis(hydroxymethyl)-2,2',2''-nitrotriethanol acetate, 200 mmol/liter, is the buffer of choice. Thioglycerol, 20 mmol/liter, is superior for both in-assay reactivation and for storage stability of sera. Fluoride, 25 mmol/liter, a broad inactivator of adenylate kinase (EC 2.7.4.3), has little effect on creatine kinase and is superior to AMP for adenylate kinase inhibition in the assay of creatine kinase. Magnesium ion, ADP, and buffer concentrations are interdependent and their optima must be determined together. The hexokinase/glucose-6-phosphate dehydrogenase activity ratio should not exceed 1.6. The range of linearity is limited by the glucose-6-phosphate dehydrogenase and NAD+ concentrations. Glucose-6-phosphate dehydrogenase, ADP, and NAD+ are the constituents most likely to result in unacceptable blanks. Creatine kinase is inhibited noncompetitively by anions: acetate and fluoride inhibit slightly, but sulfates, nitrates, and excessive chlorides should be avoided.

Evaluation of current methods for creatine kinase isoenzyme fractionation.

I have evaluated electrophoretic, ion-exchange, kinetic, selective activation, and immunological methods of fractionating creatine kinase isoenzymes for diagnostic purposes. The last three were unsuitable. Electrophoretic methods had superior resolution and reliability, but were relatively insensitive when conventional film-drying techniques were used. With elution from the film, sensitivity was comparable to that of the ion-exchange methods. Electrophoretic methods require more skill than the other methods. Ion-exchange methods had acceptable resolution and reliability with potentially superior sensitivity; they require less skill than electrophoresis, but require about as much time. Batch operation of ion-exchange yielded superior efficiency and sensitivity, and was otherwise comparable to column operation.

Creatine kinase: stability, inactivation, reactivation.

I determined the in vitro biological half-lives or decay constants for creatine kinase isoenzymes at various temperatures. Values at 37 degrees C are consistent with values reported by others in vivo, which suggests that in vivo irreversible inactivation is primarily thermal. Reversible inactivation appears to be an oxidation-reduction phenomenon. Proteins and some inactivators (urate, catecholamines) retard irreversible inactivation and preserve isoenzyme integrity. Dilution and thiols promote reversal of inactivity. Mercaptoethanol is the preferred thiol, particularly for storage and reactivation of isoenzyme MB. MB is sensitive to light and to freeze-thawing. I recommend that specimens be cooled promptly after drawing, that mercaptoethanol (10 mmol/liter) be added, and that they be stored refrigerated. Avoid prolonged exposure to light and freezing. A model of inactivation is proposed, which is based on the assumed existence of four monomer types: active, denatured, oxidized, and insulated. The model is consistent with dilution and thiol reactivation, lag phase variations, and subtype heterogeneity.

Improved batch and column separation in the assay of hemoglobin A2.

Hemoglobin A2 is a batch fractionated on a column containing diethylaminoethyl-cellulose (DE52, Whatman) with a discontinuous buffer system at pH 8.9 and 7.3. A short micro-column method is also presented, which allows separation in less than 30 min with no interference from hemoglobin S. Both methods clearly distinguish normal from thalassemia specimens, and are simpler and more rapid than previously published methods.

A single nonaqueous diazo reagent for the rapid determination of bilirubin in serum without lipemic interference.

A nonaqueous reagent for directly determining serum bilirubin with 3,3'-dimethoxydiphenyl-4,4'-tetrazonium chloride in acidified dimethylsulfoxide is described. Conjugated or unconjugated bilirubin may be determined after extraction in acidified chloroform-ethyl acetate. The procedure is accurate (r2 0.97; recovery 100% for total, 98% for extracted bilirubin) and precise (C.V. 3.0% at 65 mg bilirubin per 1. The reagent develops endpoint color at 590 nm in less than 2 min, and the developed absorbance is stable for several hours. The method is intended for special applications where gross lipemia is encountered. Standardization with pure bilirubin in dimethylsulfoxide or chloroform is identical to standardization with aqueous bilirubin in protein standards.

Improved separation of creatine kinase cardiac isoenzyme in serum by batch fractionation.

I describe a simple, single-tube batch fractionation procedure for separating MM and MB isoenzymes of creatine kinase on a macroporous strong anion exchanger (AG MP-1, Bio-Rad Laboratories). The isoenzymes can be separated in less than 3 min, with a resulting dilution of the serum with no more than an equal volume of buffer. Without sample concentration or spectrofluorometric measurement, the procedure detects 4 U of MB isoenzyme per liter. Sensitivity is limited by the sensitivity and precision of the method of measurement. The CV for the fractionation can be held to less than 4.0% at 65 U of MB per liter. Current fractionation methods are compared to the proposed procedure. With use of a discrete analyzer (Du Pont aca) the mean MB activity in a population free of heart disease was 3.2 +/- 3.0 U/liter (range, 0 to 8 U/liter). The kinetics and stability of isolated isoenzymes are reported, indicating that advisability of storing or pre-incubating samples with mercaptoethanol.

Rapid estimation of hemoglobin A2 without chromatography or electrophoresis.

Hemoglobin A2 is batch fractionated with diethylaminoethyl Bio-Gel A (Bio-Rad Laboratories) equilibrated with a tris(hydroxymethyl)aminomethane HCl buffer (pH 7.68, 8.75 mmol/liter, 6.36 mmol of CL- per liter). Hemoglobin A1 and F become bound to the resin, allowing the separation and quantitation of A1. Hemoglobin S alters the equilibrium condition, but adjustments are easily made so that A2 can be separated in the presence of S. The procedure is simpler than electrophoretic or chromatographic methods, requires 5 min, is accurate (A2 fraction is at least 94% A2, less than 6% A1), precise (SD +/-0.24 for duplicates), and has a normal limit of 2.6 +/-1.02%.