Monocyte nonspecific esterase. Enzymologic characterization of a neutral serine esterase associated with myeloid cells.

Monocytes contain a characteristic, prominent set of membrane-bound nonspecific esterases with a slightly acid isoelectric point. These esterases are also detected at modest levels in some granulocyte preparations. They are not apparent in lymphocytes. Among 18 fresh myeloid leukemias and myeloid leukemia cell lines, those of subtypes M4 (myelomonocytic) and M5 (monocytic) were strongly positive; some of subtypes M1-M3 (granulocytic) were moderately positive. The esterases were not detected among 32 fresh lymphoid leukemias and lymphoid leukemia and lymphoblast cell lines. The membrane-bound monocyte esterases, solubilized by treatment of monocyte preparations with nonionic detergent, were resolved by ion-exchange chromatography. The monocyte species account for 80-95% of the total nonspecific esterase activity of monocytes. The resolved enzymes behave as neutral serine carboxyl esterases and are highly sensitive to inhibition by diisopropylfluorophosphate (DFP) and also by sodium fluoride. Similar analysis of a lymphocyte preparation yielded no detectable monocyte esterases, but yielded numerous other forms which were generally resistant to inhibition by DFP and NaF. These nonspecific esterases are also present at background levels in monocytes. The resolution and characterization of the membrane-bound serine esterases from monocytes demonstrates the basis for the well-known cytochemistry of monocytes. The results are also crucial to the development of an immunologic surface marker test for myeloid cells and the study of monocyte membrane physiology.

Blood glutathione and cysteine concentrations in twin children.

Glutathione and cysteine are major antioxidants in blood that are associated with health and longevity. To ensure their measurement, careful attention to avoid auto-oxidation is necessary to stabilize the samples. Since no report of these compounds has been reported in children, our goal was to determine their levels of reduced and oxidized glutathione (GSH and GSSG) and cysteine (Cys and CSSC), To this end, 140 healthy children, ages 2 to 9 years from the Louisville Twin Study were studied. Blood samples were collected and analyzed for GSH, GSSG, Cys, and CSSC by our HPLC dual electrochemical method. The results showed that GSH and total GSH (GSH + GSSG) levels for monozygotic (MZ) twins were significantly higher (P < 0.001) than levels for dizygotic (DZ) twins. However, the opposite occurred for Cys and total Cys (Cys + CSSC) in that the levels were significantly higher for DZ twins than for MZ twins. (P < 0.005-0.013). In spite of this marked difference in zygosity, the within-pair correlations for twin pairs used for estimating heritability suggested that there was a major environmental influence for total GSH and total Cys. Finally. GSH levels were significantly lower for young (2-9 years) children than adults (P < 0.001).

Nonspecific esterases of the formed elements: zymograms produced by pH 9.5 polyarcrylamide gel electrophoresis.

The formed elements of human blood each contain multiple isoenzymes of nonspecific esterase that hydrolyze short chain alpha naphthyl esters. Zymograms that are characteristic of each type of formed element are obtained by subjecting purified preparations of each to polyacrylamide slab gel electrophoresis at pH 9.5 and subsequent staining of the gels for esterase activity. The most prominent isoenzyme detected is a species of low mobility that is reactive with either acetyl or butyryl esters and is highly sensitive to inhibition by 40 mM sodium fluoride. Also detected are several major acetyl esterases and a single butyryl esterase, all of which are relatively fluoride resistant. The intercellular distribution of isoenzymes varies from element-specific to pancellular. The prominent fluoride-sensitive acetyl, butyryl esterase, is the major isoenzyme of monocyte zymograms, which is consistent with the well known cytochemistry of monocytes. Lesser but significant amount (2%-3% of monocyte levels) of this isoenzyme were also detected in granulocyte zymograms. This system may prove useful in the study of differentiation of blood cells and in the classification of acute leukemias.

Purification and properties of mosquito DNA polymerase.

For the first time, DNA polymerase in a postembryonic insect has been purified and characterized. This enzyme from mosquito larvae was purified 1700-fold and was free of deoxyribonuclease and protease activities, which hindered previous investigations of insect polymerases. The enzyme had a molecular weight of 132,000 by gen filtration and aggregated to higher molecular weights when concentrated. With an activated DNA template, the pH optimum was 7.2 in phosphate buffer, and the Mg2+ concentration optimum was 5 to 10 mM. Polymerase activity was inhibited by the antisulfhydryl reagents, N-ethylmaleimide and p-mercuribenzoate, and by KCl. These properties indicate that the mosquito enzyme resembles mammalian alpha-polymerase but differs in its lack of inhibition to low ethanol concentrations. There was no evidence of a beta-polymerase form in the mosquito.

Pronounced increases in the concentration of an ovarian tumor marker, CA-125, in serum of a healthy subject during menstruation.

CA-125 is a glycoprotein associated with various ovarian tumors. A commercial radioimmunoassay involving a monoclonal antibody is available for it. In our laboratory, a normal-value study was conducted as part of a routine evaluation of this assay. One healthy subject had a serum CA-125 concentration greater than 300 kU/L, more than eightfold the upper limit of normal (35 kU/L). This increase, which coincided with the onset of the menstrual period, subsided to within the normal range by the end of the menstrual cycle. The half-life of CA-125, calculated from this decrease, was 6.4 days. Similar observations were made in the same subject over several menstrual cycles. Results of clinical and ultrasound examinations of the subject for ovarian tumors were negative. No clinical evidence of malignancy was present eight months after the initial discovery of an increased CA-125. None of the other 39 healthy subjects had a CA-125 value greater than 51 kU/L. Five of these subjects had CA-125 determined several times during their menstrual cycles; none exhibited pronounced variations in CA-125 concentrations. Evidently CA-125 can be extremely increased in a healthy woman, and possible effects of the menstrual period on serum CA-125 concentrations should be considered in pre-menopausal patients.

Nonspecific esterase of B lymphocytes from a case of chronic lymhocytic leukemia and of normal T lymphocytes: similar constellations of isoenzymes.

Lymphocytes from a case of B-cell chronic lymphocytic leukemia (CLL) were obtained in a highly purified state from a therapeutic leukapheresis preparation. The CLL lymphocytes showed a fine, scattered, granular pattern of nonspecific esterase cytochemical reactivity with either alpha-naphthyl acetate (alpha NA) or alpha-naphthyl butyrate (alpha NB) substrate as opposed to the more focal pattern of control (T) lymphocytes. Nonspecific esterase of CLL lymphocytes and normal control lymphocytes was equally resistant to inhibition by fluoride ion. Extractable nonspecific esterases from the CLL lymphocytes and from purified normal T lymphocytes were indistinguishable in regard to specific activity, substrate specificity, pH optima, and zymogram profiles on polyacrylamide gel electrophoresis at pH 9.5 and pH 4.0. Zymograms of alpha NA esterase and alpha NB esterase prepared by isoelectric focusing were also similar, with no unequivocal differences. These results are consistent with recent reports that B lymphocytes contain detectable nonspecific esterase and suggest that the B lymphocytes from this case of CLL contained a constellation of isoenzymes similar to that of normal T lymphocytes. This is interpreted as a reflection of the close kinship of these cells.

Interference of polyclonal free light chains with identification of Bence Jones proteins.

We present a case in which kappa free light chains caused difficulty in interpreting classical urinary immunoelectrophoresis, but immunofixation electrophoresis (IFE) demonstrated the presence of a lambda-Bence Jones protein. Analysis of the urine by Ouchterlony double diffusion and IFE after gel-filtration chromatography showed that the difficulty was caused by the presence of large amounts of polyclonal free light chains. The workup also demonstrated that although IFE is the more sensitive and specific technique, IFE performed on concentrated urinary samples is especially subject to misinterpretation unless densely staining patterns are diluted and reassayed. This process of sample dilution provides a means for titrating antigen and antibody concentrations such that condition-specific patterns become visible on the gel. This workup also shows that, at some dilutions, polyclonal free light chains may migrate in the same manner as an oligoclonal band in a so-called ladder configuration. These bands were observed from both monomeric and dimeric fractions isolated by gel chromatography, consistent with reports that this pattern is largely linked to the isoelectric points of the molecules. We speculate that, in rare instances, the distinction between polyclonal and monoclonal kappa free light chains migrating as a ladder-banding pattern may be equivocal.

Blood glutathione decreases in chronic diseases.

Previously a high blood glutathione level was correlated with long life span in the mouse and rat and in healthy elderly human beings. This raised the question of whether low glutathione levels occur in unhealthy subjects. To this end, 74 consecutive patients newly admitted to the hospital, with ages ranging from 21 to 89 years and diagnosed with chronic diseases, were studied along with 32 healthy control subjects. Blood samples were analyzed for reduced (GSH) and oxidized (GSSG) glutathione with a high-performance liquid chromatography-dual electrochemical method. The data were integrated with the clinical diagnoses and statistically analyzed. Marked total glutathione decreases from the control levels occurred in over 36% of the patients with chronic diseases including cancer and genitourinary, gastrointestinal, cardiovascular, and musculoskeletal diseases (P < .001). The deficit was due to low GSH concentrations and not to GSSG, which was the same as that in the control subjects. The conclusion is that a decrease in GSH is a risk factor for chronic diseases that may be used to monitor the severity and progress of the diseases. Future work is necessary to elucidate the mechanism of action.

Improved liquid-chromatographic determination of mexiletine, an antiarrhythmic drug, in plasma.

In this improved reversed-phase liquid-chromatographic procedure for determination of mexiletine in plasma, mexiletine and an internal standard, chlorodisopyramide, are extracted with methylene chloride from 0.5 mL of serum or plasma; the extract is then concentrated and injected onto a C18 chromatographic column. Mexiletine in the column effluent is detected by monitoring absorbance at 210 nm. It is quantified by use of mexiletine-internal standard peak-height ratios. The relation between this ratio and mexiletine concentration is linear from 0.1 to 5.0 mg/L. The lower limit of detection is about 50 micrograms/L. At a mexiletine concentration of 2.0 mg/L in serum, intrarun precision (CV) is 2.9% and inter-run precision is 5.9%; at 0.5 mg/L, these CVs are 5.7% and 9.6%, respectively. Analytical recovery of added mexiletine in serum is 68-88%. Therapeutic concentrations of some commonly administered drugs in patients' specimens did not interfere. In serum from 38 patients receiving mexiletine for cardiac arrhythmia, concentrations measured by this method correlated with therapeutic efficacy.