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M Levitz

Publications and source records attributed to M Levitz.

At least 37 records · Page 2Linked to original sources
PubMed1991

Purification and properties of an esterase from human breast cyst fluid.

Levels of estradiol 17 beta-ester hydrolytic activity in the breast cyst fluid (BCF) from 25 different women with fibrocystic disease of the breast were found to vary over a wide range (0-2.4 nmol/min/mg protein for estradiol acetate). On the basis of electrophoretic mobility on agarose gels, the activity from different individuals appeared to be identical. The esterase activity from a single BCF sample was purified to near homogeneity by differential ammonium sulfate precipitation, ion-exchange, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after the final purification step, showed two bands with molecular weights of approximately 22,000 and 23,000, neither of which was immunoreactive with a rabbit antibody raised to a crude esterase-free BCF preparation. Esterase activity could be demonstrated after extraction and renaturation of the protein eluted from the Mr 22,000 band. Resolution of the gel, however, was not good enough to rule out the presence of esterase activity in the Mr 23,000 protein. High performance liquid chromatography gel exclusion chromatography indicated a molecular weight of 90,000-95,000 for the esterase activity in crude BCF and approximately 225,000 for the purified activity, suggesting the native protein to be a tetramer which aggregated during purification. Although the natural substrate of the BCF esterase is unknown, the enzyme is able to cleave a variety of esters including acetate, valerate, and stearate esters of estradiol and p-nitrophenyl hexanoate. It is completely inhibited by diisopropylflurophosphate and diethylnitrophenyl phosphate and partially inhibited by NaF and ebelactone. The substrate and inhibitor profile of the enzyme indicates that it is a "B"-type carboxylesterase and not a protease. A comparison of the properties of the BCF esterase with those of esterases from the formed elements of the blood or from plasma suggests that the BCF esterase is not of blood origin and is probably derived from the cyst itself. Physiologically inactive lipoidal estrogens have been shown to be present in many human body fluids and tissues and it is possible that these esters serve as storage forms of the active hormone in hormonally sensitive tissues where the free steroid could be regenerated by hydrolysis.

Anti-Bacterial Agents↗
PubMed1991

Estradiol esters can replace 17 beta-estradiol in the stimulation of DNA and esterase synthesis by MCF-7 cells: a possible role for the estrogen-sensitive MCF-7 cell esterase.

In this communication we extend our earlier observations on estrogen-sensitive carboxyl esterases in MCF-7 human breast cancer cells able to hydrolyze esters of estradiol. Using either estradiol acetate or p-nitrophenyl hexanoate as substrates, esterase activity was found to increase 2-3-fold in MCF-7 cells maintained in the presence of 10(-8) M estradiol. Following sucrose density centrifugation, over 85% of total esterase activity was found in the cytoplasmic fraction. No esterase activity was found in spent media from growing cells. By size exclusion chromatography, estradiol acetate esterase activity exhibited a mol. wt of 45-50 kDa. Attempts to demonstrate incorporation of [3H]estradiol into estradiol fatty acid esters by the above MCF-7 cell line (203P) were unsuccessful, although, such incorporation could be demonstrated in two other MCF-7 cell sublines. Incubation of the 203P cells with 10 nM [3H]estradiol in the presence of 0.5 mM radioinert estradiol acetate resulted in the incorporation of 35 +/- 12% of the label into the estradiol acetate in 10 min. In the absence of radioinert estradiol acetate, no incorporation was observed. When MCF-7 cells were incubated with [3H]estradiol in the presence of a large excess of radioinert estradiol valerate, label was found only in estradiol valerate. Similarly, when the incubation was carried out in the presence of a mixture of radioinert estradiol acetate and valerate, label was incorporated into both esters. We conclude that the apparent formation of radiolabeled estradiol esters by MCF-7 cells incubated under the above conditions, results at least in part, from an esterase-catalyzed exchange reaction. Under conditions where no ester hydrolysis could be detected in the absence of cells, valerate and stearate esters of estradiol were found to be as effective as unesterified estradiol in stimulating esterase synthesis and the incorporation of [3H]thymidine into DNA. These results are consistent with a model in which an intracellular esterase in MCF-7 cells can generate estradiol from an exogenous lipoidal steroid and elicit an estrogen response.

Acetates↗
PubMed1990

Bile acids in human breast cyst fluid: the identification of lithocholic acid.

Breast cyst fluid (BCF) aspirated from 12 women with fibrocystic disease of the breast and sera obtained simultaneously were analyzed for bile acids. Analysis was performed by gas-liquid chromatography of the acetoxy methyl esters of the bile acids prepared after alkaline hydrolysis of the bile salts. An internal standard served to correct for methodological losses. Low levels of bile acids were found in serum samples, precluding overt hepatobiliary complications. Deoxycholic acid (17-160 mumol/L), chenodeoxycholic acid (18-305 mumol/L), and cholic acid (3-119 mumol/L) were detected in 11 of 12 samples of BCF. In 2 cases, chosen at random, the identities of the bile acids were verified by mass spectrometry. Lithocholic acid (9-23 mumol/L), a reported cocarcinogen, was detected in 6 of the 12 samples of BCF. This is the first report of the presence of lithocholic acid in BCF with confirmation by Mass spectrometry. There was no correlation between the levels of individual bile acids and those of potassium ion, Na+/K+, estriol-3-sulfate, or 16 alpha-hydroxyandrogen sulfates that had been quantified previously in these samples. There was borderline correlation between concentrations of total bile acids and K+ (P less than 0.06) and Na+/K+ (P less than 0.07). Yet to be elucidated are the mechanism of accumulation of bile acids in BCF and whether levels of particular bile acids in BCF may serve to identify that small subset of women with fibrocystic disease at risk for developing breast cancer.

Adult↗
PubMed1989

The concentration of 16x-hydroxy androgens in serum and cyst fluid of women with gross cystic disease of the breast.

The concentrations of 16 alpha-hydroxydehydroepi-androsterone sulfate (16 alpha-OHDHAS) and androst-5-ene-3 beta, 16 alpha-, 17 beta-triol-3-sulfate (A-TriolS) were measured in the plasma and breast cyst fluid (BCF) of women with gross cystic disease of the breast. In the 19 BCF samples analyzed, the 16 alpha-OHDHAS and A-TriolS concentrations ranged from 15 to 1130 ng/mL, and 12 to 871 ng/mL, respectively. However the concentrations of these steroids in the sera of these women were lower (15-179 ng/mL, 8-80 ng/mL, respectively). Estriol-3-sulfate (E3-3S) concentrations in the BCF samples ranged from barely detectable (0.2 ng/mL) to 3 micrograms/mL. In BCF or serum a positive linear correlation was observed in the concentration of 16 alpha-OHDHAS and A-TriolS (p less than 0.001 and 0.05, respectively). However, in the same patients no statistical significance was observed in the BCF vs serum concentrations of these two steroids. When the specimens from this and previous studies were combined, positive correlation was found between potassium ion concentration and E3-3S or 16 alpha-OHDHAS. The origin of the high concentration of E3-3S is still obscure. Although no linear correlation between 16 alpha-OHDHAS and E3-3S was observed, the possibility of a precursor-product relationship between the two is not elimnated.

Androgens↗
PubMed1988

Placental transfer of thiamine in the human subject: in vitro perfusion studies and maternal-cord plasma concentrations.

The transfer of thiamine across human placenta has been studied in an in vitro perfusion system. With the maternal and fetal perfusates not recirculated, the transfer index (ratio of clearance of thiamine to that of L-glucose) toward the fetus was 2.09 +/- 0.36, suggesting a mediated transport system. In the reverse direction, the transfer rate approximates that expected from simple diffusion (transfer index 0.78 +/- 0.25). Stepwise increases in thiamine concentration in the maternal perfusate were associated with parallel increases in transfer rates up to 500 nmol/L. Above that concentration, the rate of increase declined and the transfer index fell. When the maternal perfusate was recirculated and the fetal circulation was kept open, the thiamine concentration rapidly decreased, reaching a plateau in 30 to 40 minutes at a level about 0.1 to 0.2 of the fetal perfusate. In contrast, the concentration within the placenta greatly exceeded those in the perfusates. The observations indicate an effective transport system directed toward the fetus, saturable at low concentrations and capable of establishing a transplacental gradient. Measurement of thiamine concentrations in 20 paired samples of plasma obtained at term demonstrated a mean (+/- SD) cord:maternal ratio of 2.5 +/- 1.4. Differential protein binding was excluded as a cause of the gradient. The in vivo gradient is probably attributable to placental transport.

Blood Proteins↗
PubMed1988

Placental transport of riboflavin: differential rates of uptake at the maternal and fetal surfaces of the perfused human placenta.

The kinetics of riboflavin transfer across the perfused human placenta has been studied. With both circulations open, the transfer index (clearance of riboflavin: clearance of antipyrine) toward the fetus was 3.3 +/- 0.08 (+/- SD) versus 0.87 +/- 0.02 in the reverse direction (p less than 0.01). Galactoflavin reduced the materno-fetal transfer rate. The uptake of riboflavin and L-glucose after bolus injection into the maternal perfusate was 35% +/- 9.6% versus 3% +/- 16%, p = 0.005, which indicates an uptake rate in excess of diffusion. There was no significant difference in uptake at the fetal surface. With recirculation of either the maternal or fetal perfusate, the transplacental gradient that was established was lower at high concentrations of riboflavin, which indicates the saturability of this function. Riboflavin was concentrated in the intracellular space and partly metabolized to flavin mononucleotide and flavin adenine dinucleotide. An equilibrium was approximated when both perfusates were recirculated, with the concentration lowest in the maternal perfusate, highest in the placenta, and intermediate in the fetal perfusate. It is concluded that the major factor directing riboflavin toward the fetus is the difference in rates of uptake at the two surfaces of the placenta.

Biological Transport↗
PubMed1987

An estrogen-dependent esterase activity in MCF-7 cells.

The presence of steroidal esters in hormonally sensitive tissues lends importance to the esterases which convert the biologically inactive adducts to the parent potent forms. Accordingly, esterase-activities were studied in a human breast cancer model--the MCF-7 cell line. Tritiated estradiol esters- estradiol-17-acetate (EA), estradiol-17-valerate (EV) and estradiol-17-stearate (ES) were tested systematically, but 3 beta-ol esters of androgens, and phorbol diesters were also investigated. All compounds tested, except the phorbol diesters were hydrolyzed either when added to growing cultures or to the 28,000 g supernate of homogenized MCF-7 cells. Among the estrogens, the relative rates of hydrolysis were EA greater than EV greater than ES. The esterase for EA was different as it was not inhibited by saturating concentrations of EV or ES, and unlike the others its activity was stimulated by the addition of estradiol to the culture medium. The antiestrogen keoxifene,[(6-Hydroxy-2-(4-hydroxyphenyl)benzo less than b greater than thien-3-yl greater than less than 4- less than 2-(1-piperidinyl)ethoxy greater than phenyl greater than methanone], negated the stimulatory effect. Other major classes of steroids did not influence EA esterase activity. Results of inhibition experiments indicated that the esterases are of the serine active-site types. The significance of the estrogen-dependent esterase activity can be assessed when the natural substrate(s) for the enzyme is elucidated.

Breast Neoplasms↗
PubMed1987

Riboflavin concentration in maternal and cord blood in human pregnancy.

Riboflavin concentration was measured in sera of a control population and in a series of paired maternal and cord sera. The assay technique was carefully validated and appears to be specific and reproducible. The mean riboflavin concentration in 12 apparently healthy adults was 116 +/- 46 nmol/L (SD). In 20 uneventful pregnancies the cord serum concentration was generally higher than the maternal concentration (158 +/- 47 nmol/L versus 113 +/- 35 nmol/L; p = 0.001). The cord-to-maternal ratio in paired sera averaged 1.45 +/- 0.44. There was no detectable difference in binding of riboflavin to cord and maternal serum proteins as measured by equilibrium dialysis (59.0% +/- 17% versus 60.8% +/- 16%). Comparison of protein binding by paired cord and maternal sera yielded a ratio of 0.99 +/- 0.13. The transplacental gradient of riboflavin concentration is unrelated to protein binding and is consistent with active transport by the placenta, as previously demonstrated in vitro.

Blood Proteins↗
PubMed1986

Establishment of gradients of riboflavin, L-lysine and alpha-aminoisobutyric acid across the perfused human placenta.

The mechanism for establishing transplacental gradients was studied in the dually perfused human placental lobule by recirculating either the maternal or fetal perfusate and leaving the other circuit open. Antipyrine, used as a reference molecule, rapidly reached equivalent concentrations in both perfusates regardless of which was recirculated. In contrast, riboflavin, L-lysine and alpha-aminoisobutyric acid showed distinctive kinetics at each surface of the placenta. Recirculation of the maternal perfusate quickly depressed the concentrations of these compounds and maintained them at levels of 50-70% of the fetal concentration. Recirculation of the fetal perfusate was followed by a gradual increase in concentrations, establishing gradients 1.2-1.7 times the maternal. It appears that uptake at the maternal surface greatly exceeds release, whereas the two rates are more nearly balanced at the fetal surface. The net effect is extraction of substrate from the maternal circulation and transfer to the fetal, where it accumulates to form a gradient.

Aminoisobutyric Acids↗
PubMed1985

The 65-kDa phorbol-diester hydrolase in mouse plasma is esterase 1 and is immunologically distinct from the 56-kDa phorbol-diester hydrolase in mouse liver.

Esterase 1, a well-characterized mouse plasma protein of unknown function, has activity against a wide range of ester substrates including beta-alanine nitrophenyl esters and 17 beta-esters of estradiol. In this article, we report that esterase 1 is also responsible for a majority of the phorbol-12-ester hydrolase activity in mouse plasma. Incubation of homogeneous esterase 1 with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) at either 4 or 37 degrees C for up to 18 h yielded phorbol 13 alpha-acetate as the only hydrolysis product. Specific polyclonal antibodies to esterase 1 inhibited 95% of PMA hydrolysis by a purified esterase 1 preparation and 65% of PMA hydrolysis by mouse plasma. Perfused mouse liver homogenates contain two distinct phorbol diester hydrolases with apparent molecular masses of 65 kDa and 56 kDa, respectively. The 65-kDa protein appears to be immunologically identical to the plasma enzyme, while the 56-kDa protein, found in liver but not in plasma, is immunologically distinct. Phorbol 12-myristate, phorbol 12,13-dibutyrate, and PMA were found to be competitive inhibitors of the beta-alanine-nitrophenyl esterase activity of esterase 1 with Ki values of approximately 7 microM. Phorbol 13-acetate and phorbol itself were less effective with Ki values of 37 and 140 microM, respectively. Sodium salts of valeric and myristic acids did not inhibit at 10 microM. The above results indicate that efficient substrate binding requires a phorbol 12-ester. Similar results were obtained with estradiol 17 beta-valerate which is a better substrate for esterase 1 than is PMA. Our results strongly suggest that esterase 1 and a recently described phorbol ester hydrolase isolated from mouse serum (Saito, M., and Egawa, K. (1984) J. Biol. Chem. 259, 5821-5826) are the same and are immunologically and kinetically distinct from the 56-kDa phorbol 12-ester hydrolase in mouse liver.

Animals↗
PubMed1985

On the stability of salivary progesterone under various conditions of storage.

The concentrations of progesterone in saliva of women exhibited significant decreases when the fluid was stored in plastic vials for 3 days at room temperature or 37 C. The addition of antibiotics or a variety of metabolic poisons to the saliva prior to storage did not prevent the progesterone decrement. However, the addition of albumin (2 g/dl) was protective, suggesting that the protein impeded adsorption of salivary progesterone by the plastic container. Saliva could be maintained at 37 C for 3 days in glass vials or at -20 C in plastic containers for indefinite periods without loss of progesterone titers. These data indicate that a patient under luteal function assessment may collect saliva samples in glass vials at regular intervals during the latter half of her cycle and store them in the freezer compartment of the refrigerator until shipment by mail to the laboratory for progesterone assay. With special care, plastic vials charged with albumin may also be used.

Albumins↗
PubMed1985

Transfer of riboflavin by the perfused human placenta.

The transfer of radioactive riboflavin across the human term placenta has been studied in an in vitro perfusion system. The clearance index (clearance riboflavin:clearance antipyrine) toward the fetus averaged 0.69 and the transfer index (clearance riboflavin:clearance L-glucose) averaged 3.40. The respective indices in the reverse direction were 0.25 and 0.87. Stepwise increases in the concentration of riboflavin in the maternal perfusate were associated with parallel increases in transfer rates, expressed as ng/min, up to concentrations approximating 100 ng/ml. Above that concentration, the transfer rates continued to increase at a slower rate. Concurrently, there was a reduction of the transfer index from 2.7 to 1.1 at 1000 ng/ml. With the fetal circulation closed, the placenta established a gradient toward the fetus over a period of 150 min of 1.7. The transferred radioactivity was identified as riboflavin by high-performance liquid chromatography, whereas that retained within the placenta was metabolized to flavin mononucleotide (33-75%). The observations indicate a very effective active transport system directed toward the fetus which is limited in capacity to low concentrations of riboflavin.

Antipyrine↗
PubMed1985

Androsterone long chain fatty acid esters in human breast cyst fluid.

We reported previously that incubation of [3H] androsterone in homogenates of human breast tumor resulted in production of long chain fatty acid esters of androsterone (A-LCFE). To identify the individual A-LCFE, breast tumor homogenates were incubated with androsterone, then submitted to solvent extraction, Celite chromatography, high pressure liquid chromatography and OH- negative chemical ionization mass spectrometry. The (M-1)-ions of the oleate, linoleate, palmitoleate, palmitate, arachidonate, and stearate esters of androsterone were produced. The first 3 cited unsaturated esters accounted for over 90% of the total. Since fibrocystic disease of the breast is a reported risk factor for the development of breast cancer, breast cyst fluids were analyzed for A-LCFE as part of an overall program to relate endocrine profiles in cyst fluid to the incidence of cancer. Breast cyst fluids were analyzed for total A-LCFE by a method involving solvent extraction, saponification, purification of the liberated androsterone, and then quantification of the steroid by RIA. The 10 fluids analyzed contained 0.52-3.79 ng/ml fatty acid esters, measured as androsterone. In 4 of these samples, the individual A-LCFE were analyzed by mass spectrometry. As in the incubation study, the unsaturated fatty acid esters predominated. In 3 samples, palmitoleate and in 1 sample, oleate predominated. The palmitate varied from undetectable to 25% of the total. The divergent total concentrations and profiles of A-LCFE indicate potential parameters for correlations with the subsequent course of fibrocystic disease of the breast.

Androsterone↗
PubMed1984

Transfer of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 across the perfused human placenta.

The rates of placental transfer of 25-hydroxyvitamin D3 [25-(OH)D3] and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] were determined in an in vitro perfusion system. Antipyrine was included in each perfusion, and the data are expressed as clearance index, the ratio of hydroxyvitamin D3 to antipyrine clearance. In most experiments, serum, 0.2%, was added to the perfusates as a source of vitamin D3-binding protein. Binding as measured by dextran-coated charcoal assay for 25-(OH)D3 was over 90%, for 1,25-(OH)2D3, only 25% to 50%. The clearance index from the maternal to fetal circulation averaged 0.02 and 0.26 for 25-(OH)D3 and 1,25-(OH)2D3, respectively. When vitamin D3-binding protein was omitted from the perfusate, the clearance indices of 25-(OH)D3 were 0.12 and 0.46 in two experiments. Binding to vitamin D3-binding protein is a major determining factor for the transfer rates of 25-(OH)D3 and 1,25-(OH)2D3.

Antipyrine↗
PubMed1984

Intermediary metabolism of estriol in pregnancy.

Estriol (E3), the most abundant estrogen in pregnancy is produced predominantly in the placenta from androgen precursors of fetal origin. The estriol so formed is secreted efficiently into the maternal circulation where it is converted to 4 conjugates--estriol-3-sulfate (E3-3S), estriol-16-glucosiduronate (E3- 16G ), estriol-3-glucosiduronate (E3- 3G ) and estriol-3-sulfate-16-glucosiduronate (E3-SG). The order of renal clearances is E3- 16G greater than E3- 3G greater than E3-3S approximately E3-SG. Unconjugated E3 and E3- 3G differ from the other forms of estriol in that their removal from the blood compartment is essentially irreversible. E3-3S, E3- 16G and E3-SG undergo interconversions during enterohepatic circulation and eventual partial conversion to E3- 3G . Following delivery of the fetus and placenta, unconjugated E3 is no longer detectable in the maternal serum within 1-2 h, whereas the concentrations of the conjugates decline more slowly, the rates being determined by the rates of renal clearance and enterohepatic interconversions. E3- 3G levels were dramatically elevated in a case of Group C polycystic kidney disease, providing evidence that this conjugate is indeed an end-product of estriol metabolism.

Cholestasis, Intrahepatic↗
PubMed1984

Estriol in human breast cyst fluid.

Although cystic lesions of the breast are not precancerous per se, statistical studies have indicated that this condition predisposes a 2- to 4-fold greater risk for breast cancer. Seeking a hormonal etiology to this correlation, investigators have analyzed breast cyst fluid ( BCF ) for steroids and have compared the levels to those in the blood. The 17-ketosteroids-androsterone, dehydroisoandrosterone and their sulfates are elevated in BCF . The same is true for estrone sulfate and estradiol-3-sulfate. We have found the most dramatic differences with estriol-3-sulfate (E3-3S), the concentration of which ranged from 187-6134 pg/ml in over 40 specimens analyzed, whereas in 12 serum specimens from normal women, E3-3S was barely detectable. The origin of E3-3S is not known. [3H]E3-3S is not concentrated in BCF following its injection into an arm vein. The blood half-life of [3H]E3-3S is much lower than that of estrone sulfate. Samples of breast nipple aspirates from normal women were also analyzed for E3-3S. None could be detected. The best explanation of the data accumulated thus far is that E3-3S is synthesized at the epithelial lining of the cyst and released into the BCF , from which its efflux is inefficient.

Adult↗