Intravenous administration of irinotecan elevates the blood beta-glucuronidase activity in rats.

7-Ethyl-10-hydroxycamptothecin (SN-38) is the active metabolite of an anticancer drug, irinotecan (CPT-11). Severe late diarrhea is the dose-limiting toxic effect of CPT-11. This diarrhea has been examined regarding biliary excretion and deconjugation of SN-38 glucuronide by the enzyme beta-glucuronidase (beta-GL) in intestinal microflora. Prompted by the enzymological and structural similarity of CPT-11 to organophosphorus and carbamate insecticides, we studied the effect of CPT-11 on blood beta-GL activity in rats. The i.v. injection of CPT-11 in rats significantly elevated their plasma beta-GL activity (with phenolphthalein glucuronide as a substrate) at doses of 10 and 40 mg/kg, with peak activity observed 2-3 h after administration. SN-38 lactone and carboxylate had no effect on the plasma beta-GL level. The enhancement of the activity was also observed in serum using SN-38 glucuronide as a substrate. The serum beta-GL levels showed a close correlation between these substrates. The enhancement of plasma (serum) beta-GL activity is suggested to be a result of the release of beta-GL from liver microsomes. Serum and microsomal carboxylesterase were not significantly affected by CPT-11 administration.

Inhibitory effect of phloridzin and phloretin on glucuronidation of p-nitrophenol, acetaminophen and 1-naphthol: kinetic demonstration of the influence of glucuronidation metabolism on intestinal absorption in rats.

Intestinal glucuronidation and absorption of p-nitrophenol (p-NP), acetaminophen (APAP) and 1-naphthol (alpha-NA) in the presence of phloridzin (inhibitor of Na+/glucose cotransporter) and phloretin (aglycone of phloridzin) were studied. Glucuronides of p-NP, APAP and alpha-NA appeared on both the serosal and mucosal sides. The amounts of glucuronides on the serosal side were decreased in the presence of phloridzin and phloretin. p-NP, APAP and alpha-NA appeared on the serosal side as well, and the amounts of p-NP, APAP and alpha-NA on the serosal side were increased by the presence of phloridzin and phloretin. Furthermore, the intestinal glucuronidation and absorption of alpha-NA at various concentrations were studied in the presence and absence of phloretin. Metabolic clearance was decreased in the presence of phloretin, and the absorption clearance was increased. The higher concentrations of alpha-NA caused higher absorption clearance. The lower the metabolic clearance, the higher the absorption clearance. The relationship between glucuronidation metabolism and absorption in intestine was kinetically analyzed by the metabolic inhibition model. Complete inhibition of glucuronidation improved the intestinal absorption of alpha-NA, and the absorption clearance increased to 7.17 microliter/min/cm. The formation of phloretin and an unknown metabolite from phloridzin were observed. An unknown metabolite from phloretin was observed, and was suppressed by the presence of alpha-NA. This suggests that phloridzin was hydrolyzed to phloretin, which was metabolized to glucuronide, and thereby inhibited glucuronidation of p-NP, APAP and alpha-NA.

Intestinal Na+/glucose cotransporter-mediated transport of glucose conjugate formed from disaccharide conjugate.

Intestinal absorption of beta-disaccharide (cellobiose, maltose and lactose) conjugates of p-nitrophenol (p-nitrophenyl beta-disaccharide) were examined in terms of the hydrolysis of disaccharide conjugate to monosaccharide conjugate and the transport of monosaccharide conjugate by Na+/glucose transport carrier (SGLT1). beta-Cellobioside, beta-maltoside and beta-lactoside of p-nitrophenol (p-NP) were hydrolyzed to p-nitrophenyl beta-glucoside (p-NPbeta glc) on the mucosal side, and p-NPbeta glc appeared on the serosal side. Although p-NP beta-disaccharide, p-NP and p-NP glucuronide also appeared on the serosal side, their amounts were much lower than that of p-NPbeta glc. The amount of p-NPbeta glc transported to the serosal side was decreased in the presence of phloridzin (transport inhibitor of SGLT1) and in the absence of Na+ (a cosubstrate of SGLT1), indicating that p-NPbeta glc was formed from p-NP beta-disaccharide on the mucosal side and transported to the serosal side by SGLT1. Furthermore, the absorption clearance of p-NPbeta glc, which was formed from p-NP beta-cellobioside and p-NP beta-lactoside by lactase-phloridzin hydrolase (LPH), was much higher than that of p-NPbeta glc itself, although the absorption clearance of p-NPbeta glc, which was formed from p-NP beta-maltoside by maltase was similar to that of p-NPbeta glc itself. These results indicated that p-NPbeta glc was transported by the vectorial cooperation of SGLT1 with LPH from mucosal p-NP beta-cellobioside or p-NP beta-lactoside.

The beta-anomeric and glucose preferences of glucose transport carrier for intestinal active absorption of monosaccharide conjugates.

The anomeric preference for intestinal absorption of glucosides and galactosides (1- and 2-naphthyl glycosides) was studied by the everted rat intestine method. After the absorption of beta-glucoside and beta- and alpha-galactosides, the glycosides itself appeared on the serosal side, whereas after the alpha-glucoside absorption, the glucoside itself was not detected on the serosal side, but a large amount of aglycone appeared instead. This indicates an alpha-anomeric preference of desglucosylation through the intestinal membrane. A significant decrease of the total (glycoside + aglycone + glucuronide metabolites) amount transported to the serosal side in the absence of Na+, a cosubstrate of the glucose transport carrier (GTC), was observed in alpha- and beta-glucosides and beta-galactoside, but not in alpha-galactoside. This indicates the poor contribution of GTC to the alpha-galactoside absorption. The Na(+)-dependent absorption of the glycosides by the GTC were beta-glucoside > alpha-glucoside > beta-galactoside (for 2-naphthyl glycosides), and beta-galactoside > alpha-galactoside (for 1-naphthhyl glycosides). These results and those of a previous study led to the conclusion that the intestinal glucose transport carrier prefers beta-anomer to alpha-anomer, and glucose to galactose for monosaccharide conjugates absorption.

Intestinal transport and metabolism of analgesic dipeptide, kyotorphin: rate-limiting factor in intestinal absorption of peptide as drug.

Intestinal transport and metabolism of kyotorphin (KTP) were studied in rat everted small intestine. KTP on the mucosal side was metabolized completely within 60 min, and any amounts of KTP were not detected on the serosal side. On the other hand, [D-Arg2]-KTP (D-KTP) was stable on the mucosal side to appear on the serosal side. However, N-t-butoxycarbonyl-KTP (Boc-KTP), which was metabolized on the mucosal side faster than KTP, appeared on the serosal side. In intestinal homogenate, KTP was metabolized, and the metabolic clearance (CL(met)) was decreased by peptidase inhibitors, bestatin, o-phenanthrolin and tryptophan hydroxamate. In the presence of these peptidase inhibitors, the absorption clearance (CL(abs)) of KTP was increased. The less the CL(met) of KTP was, the more the CL(abs) of KTP was. Meanwhile, Boc-KTP in intestinal homogenate was stable even in the absence of peptidase inhibitors. The CL(abs) of Boc-KTP was constant irrespective of the stability on the mucosal side. Kinetic analysis by the metabolic inhibition model indicated that the stabilization of KTP in the intestinal tissue could increase the CL(abs) up to 0.247 microl/min per cm, which was as much as the CL(abs) of stable D-KTP. These results led to the conclusion that rate-limiting process in intestinal absorption of KTP is metabolic degradation in intestinal tissue during the absorption.

Intestinal transport and metabolism of glucose-conjugated kyotorphin and cyclic kyotorphin: metabolic degradation is crucial to intestinal absorption of peptide drugs.

Intestinal transport and metabolism of modified kyotorphin (KTP) were studied in rats. Modified KTPs studied were C-terminally modified KTP with p-aminophenyl-beta-D-glucoside (KTP-pAPbeta glc), N-terminally modified KTP-pAPbeta glc with t-butyloxycarbonyl group (Boc-KTP-pAPbeta glc) and the N- and C-terminally modified KTP by cyclization (cyclic KTP). KTP-pAPbeta glc was metabolized at a similar rate to that of KTP, and did not appear on the serosal side. Although Boc-KTP-pAPbeta glc was also metabolized, it was more stable than KTP and appeared on the serosal side. Cyclic KTP was also quite stable and appeared on the serosal side. The modified KTPs were evaluated kinetically for absorption consisting of membrane transport and metabolism. Absorption clearance (CL(abs)) of cyclic KTP, Boc-KTP-pAPbeta glc and Boc-KTP was higher than that of KTP (0.247 microl/min/cm) (Mizuma et al., Biochim. Biophys. Acta 1335 (1997) 111-119), which is the theoretical maximum by complete inhibition of peptidase activity, indicating that derivatization of KTP increases the membrane permeability. Furthermore, the data clearly showed that the greater the metabolic clearance (CL(met)) of KTP and the KTP derivatives, the lower the absorption clearance (CL(abs)). These results and further simulation study led to the conclusion that metabolic degradation in the intestinal tissues is more critical than membrane permeability (transport) for oral delivery of peptide drugs. Based on the stability of cyclic KTP in serum, this appears to be a good candidate analgesic peptide drug.

Influence of the N-B transition of human serum albumin on the structure of the warfarin-binding site.

The fluorescence quantum yield of warfarin increased with the viscosity of the medium and showed good correlation with it. The internal rotation of the acetonylbenzyl group of a warfarin molecule may thus possibly decrease in a viscous medium. the fluorescence quantum yield of warfarin bound to human serum albumin increased with the pH of the medium in the pH range of 6.2-9.0. Fluorescence-emission maximum wavelengths of warfarin bound to human serum albumin indicated a small blue-shift with the pH of the medium and that of free warfarin in the absence of albumin also shifted slightly to a shorter wavelength with the viscosity of the medium. Warfarin is bound more strongly to human serum albumin at basic pH than at neutral pH, and the increase in the bound fraction of warfarin correlated well with the increase in the fluorescence quantum yield of bound warfarin in the same pH range. Thus, the structure of the warfarin-binding site in the B (base) form appears more spatially confined than that in the N (neutral) form. The motion of the warfarin molecule bound to its binding site on human serum albumin in the N-B transition may thus be more restricted at basic than at neutral pH, and this may possibly be the reason for the stronger binding of warfarin to human serum albumin in the B form.

Human urine deoxyribonuclease II (DNase II) isoenzymes: a novel immunoaffinity purification, biochemical multiplicity, genetic heterogeneity and broad distribution among tissues and body fluids.

Deoxyribonuclease II (DNase II) was purified from the urine of a 48-year-old male (a single individual) using a column chromatography series, including concanavalin A-agarose and an immunoaffinity column utilizing anti-human spleen DNase II antibody, and was then characterized. Based on the catalytic properties of the purified enzyme, we have devised a technique of isoelectric focusing by thin-layer polyacrylamide gel electrophoresis (IEF-PAGE) combined with a specific zymogram method, for investigating the possible molecular heterogeneity of human DNase II. DNase II in urine as well as the purified form was found to exist in multiple forms with different pI values separable by IEF-PAGE within a pH range of 5-7. Since sialidase treatment of the urine sample induced simplification of the isoenzyme patterns with diminishment of anodal bands, it was clear that the multiplicity of the enzyme was in part due to differences in the sialic acid content. On screening of DNase II isoenzyme patterns in urine samples from more than 200 Japanese individuals, only the common isoenzyme pattern was observed and no electrophoretic variations were detected. However, genetic studies of urinary enzyme activity and comparative studies on the activity in urine, semen and leukocytes from the same individuals suggest that the enzyme activity level of DNase II may be under genetic control. The enzyme was widely distributed in human tissues and showed high activities in secretory body fluids such as breast milk, saliva, semen and urine, and leukocyte lysates.

Factors that cause the beta-anomeric preference of Na+/glucose cotransporter for intestinal transport of monosaccharide conjugates.

The intestinal transport of glucose- and galactose-conjugated acetaminophen (APAP glycoside) by Na+/glucose cotransporter (SGLT1) was studied. SGLT1-mediated transport of APAP glycosides preferred glucoside>galactoside and beta-anomer>alpha-anomer. These preferences agree with previous studies. NMR spectroscopic and molecular modeling studies indicated that the conformation of the glucose ring of alpha- and beta-glucosides of APAP, as well as glycosides in previous studies, is in the 4C1 chair form, the same form as glucose itself. Molecular dynamics analysis also indicated that the glucose ring was in the 4C1 chair form, and that there are differences between the rotational spaces of aglycones and hydroxy groups of glucose moieties between anomers. Therefore, we conclude that the beta-anomeric preference of glucose conjugate transport by SGLT1 is not due to the conformation of the glucose ring, but to the configuration of the aglycone at C-1 of the monosaccharide moiety.

Transport mechanisms of a glycoside, p-nitrophenyl-beta-D-glucopyranoside, across rat small intestinal brush-border membranes.

We examined the mechanism of p-nitrophenyl-beta-d-glucopyranoside (p-NP-beta-d-Glc) transport in brush-border membrane vesicles from rat small intestine. The initial uptake rate showed an overshoot phenomenon in the presence of an inwardly directed sodium-ion concentration gradient. The overshoot disappeared when the sodium-ion concentration gradient was replaced with a potassium ion concentration gradient. d-Glucose and p-NP-beta-D-Glc analogues inhibited the uptake, whereas uridine, leucine and disaccharide did not. Data on the concentration dependence of p-NP-beta-D-Glc uptake indicated that two carrier-mediated systems are involved. The uptake via the high-affinity site required an inwardly directed sodium-ion concentration gradient, while the uptake via the low-affinity site proceeded such a gradient. D-Glucose competitively inhibited the initial uptake of p-NP-beta-D-Glc via the high-affinity site with a Ki value of 301 microM. The p-NP-beta-D-Glc is transported in the small intestine via both the same carrier-mediated transport system that takes up D-glucose and a distinct low-affinity carrier-mediated transport system.

Lipid peroxidation in rat liver microsomes during naproxen metabolism.

Naproxen, a non-steroidal anti-inflammatory drug, is known to be highly effective and relatively safe, but some side-effects in the liver have been reported. In the present study, the effect of naproxen metabolism on rat liver microsomes was studied by determining lipid peroxidation in terms of thiobarbituric acid reactive substances (TBA-RS), high molecular weight protein aggregates and fluorescent substances formed in the microsomal suspension containing naproxen, NADPH and MgCl2. Lipid peroxidation was found to occur at 10 mM naproxen. Production of chemiluminescence from the microsomal suspension was observed during naproxen metabolism. The time course of 6-demethyl-naproxen formation by O-demethylation of naproxen appeared to be comparable to that of the chemiluminescence production in their initial periods of production. These results suggest that the lipid peroxidation was provoked through the reactive oxygen species generated during the oxidative metabolism of naproxen.

Chemiluminescence associated with doxorubicin-induced lipid peroxidation in rat heart mitochondria.

Chemiluminescence was observed in a rat heart mitochondrial suspension containing NADH, FeC1(3) and doxorubicin (Adriamycin) (DXR). There was good correlation between the total intensity of chemiluminescence and the total amount of thiobarbituric acid reactive substances (TBARS) produced during DXR redox cycling. Thus, the chemiluminescence was shown to be associated with lipid peroxidation. The chemiluminescence was quenched by superoxide dismutase (SOD), suggesting that superoxide anion radicals contributed to its production. Upon addition of 1,4-diazabicyclo[2,2,2]-octane (DABCO), a singlet oxygen emission enhancer, to the mitochondrial suspension emitting the chemiluminescence, the chemiluminescence intensity increased transiently, indicating the involvement of singlet oxygen. Furthermore, spectral analysis of the chemiluminescence showed it to be due to singlet oxygen and excited carbonyls.

Oxidative stress in isolated rat hepatocytes during naproxen metabolism.

Naproxen, a non-steroidal anti-inflammatory drug, induced lipid peroxidation in isolated hepatocytes of rats. The viability of the hepatocytes decreased upon lipid peroxidation, and this effect was accompanied by the formation of high molecular weight protein aggregates in the hepatocytes. Protein aggregation occurred slowly compared with the formation of thiobarbituric acid reactive substances (TBARS). The increase of TBARS was strongly correlated with the decrease of intracellular glutathione. Chemiluminescence was produced from the hepatocyte suspension during naproxen metabolism, and was correlated with the formation of TBARS. These results indicate that lipid peroxidation in the hepatocytes was provoked by reactive oxygens produced in the process of naproxen metabolism.

Evaluation of adriamycin-induced lipid peroxidation.

Lipid peroxidation is known to be a mechanism for Adriamycin-induced toxicity. In the present study, two methods which detect fluorescent substances and high molecular weight protein aggregates in peroxidized membranes were applied to Adriamycin-induced lipid peroxidation in liver microsomes. A rat liver microsomal suspension containing an NADPH-generating system was incubated with Adriamycin. Thiobarbituric acid reactive substances (TBA-RS), formed during this incubation, were transferred from the microsomes to the medium. Fluorescent substances determined by the fluorescence emitted from both the microsomes themselves and the chloroform/methanol extracts of the microsomes, were found to be formed during this incubation. High molecular weight protein aggregates determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were also formed. Fluorescent substances and high molecular weight protein aggregates were found in microsomal membranes themselves and increased time dependently. These substances retained in membranes can be of great use to delineate the site of Adriamycin-induced lipid peroxidation in vitro and in vivo and to determine how this lipid peroxidation affects the membrane.

Comparative study of active absorption by the intestine and disposition of anomers of sugar-conjugated compounds.

Active absorption in the intestine and metabolism of the beta- and alpha-anomers of the glucoside and galactoside of p-nitrophenol (p-NP) were studied to find a more suitable prodrug for poorly absorbed drugs. The everted sac technique was used to investigate the intestinal absorption of these glycosides at 250 microM from the mucosal to the serosal side in the rat jejunum. The absorption clearance of p-nitrophenyl alpha-D-glucopyranoside (p-NP alpha glc) (0.271 +/- 0.089 microL/min/cm, mean +/- SE, N = 8) was much lower than that of p-nitrophenyl beta-D-glucopyranoside (p-NP beta glc) (4.45 +/- 0.34 microL/min/cm, mean +/- SE, N = 4) which is actively absorbed by a glucose transport carrier [Mizuma et al., Biochem Pharmacol 43: 2037-2039, 1992]. However, the major constituent appearing on the serosal side was p-NP (aglycone) after absorption of pNP alpha glc, whereas it was p-NP beta glc itself after absorption of p-NP beta glc. The total amount transported to the serosal side after 20 min of p-NP alpha glc absorption, which was similar to that of p-NP beta glc, was significantly decreased in the absence of Na+, indicating the active absorption of p-NP alpha glc by a Na(+)-dependent glucose transport carrier. Perfusion with a mucosal solution of p-NP alpha glc showed that the p-NP concentration on the serosal side (15.8 +/- 1.56 microM, mean +/- SE, N = 3) was significantly (P < 0.05) higher than that on the mucosal side (5.84 +/- 1.24 microM, mean +/- SE, N = 3) at 20 min. This indicated that the p-NP appearing on the serosal side was derived not from absorption of p-NP but from hydrolysis of p-NP alpha glc through the intestinal membrane during absorption. On the other hand, after absorption of p-nitrophenyl beta-D-galactopyranoside (p-NP beta gal), which is actively absorbed by glucose transport carrier, p-NP beta gal itself appeared mostly on the serosal side. However, p-nitrophenyl alpha-D-galactopyranoside (p-NP alpha gal) absorption, which resulted in appearance on the serosal side, was not significantly decreased in the presence of 1 mM phloridzin or in the absence of Na+, indicating that the contribution of the glucose transport carrier to p-NP alpha gal absorption was minimal. The order of the Na(+)-dependent intestinal absorption was p-NP beta glc > p-NP alpha glc > p-NP beta gal > p-NP alpha gal.

Intestinal active absorption of sugar-conjugated compounds by glucose transport system: implication of improvement of poorly absorbable drugs.

The intestinal absorption of glucose- and galactose-conjugated compounds was studied in the everted sac of the rat small intestine. The absorption clearance of p-nitrophenyl beta-D-glucopyranoside (p-NPglc) at 250 microM in the mucosal side (4.45 +/- 0.34 microL/min/cm, mean +/- SE, N = 4), calculated by dividing the absorption rate by the drug concentration, was significantly decreased (0.476 +/- 0.036 microL/min/cm) in the presence of 1 mM phloridzin, an inhibitor of glucose transport, and in the absence of Na+, a cosubstrate of the glucose transport carrier (0.424 +/- 0.018 microL/min/cm). The absorption clearance of p-NPglc was decreased as its concentration increased. In the same experiment, the absorption clearance of p-nitrophenyl beta-D-galactopyranoside (1.99 +/- 0.23 microL/min/cm) was also significantly decreased in the presence of phloridzin and in the absence of Na+. However, the absorption clearance of p-nitrophenyl beta-D-mannopyranoside (0.811 +/- 0.013 microL/min/cm) was low and not significantly decreased in the presence of phloridzin (P greater than 0.1). Furthermore, the absorption clearance of beta-naphthyl beta-D-glucopyranoside and beta-naphthyl beta-D-galactopyranoside was also significantly decreased in the presence of phloridzin (P less than 0.001). These results indicated that the glucose and galactose moieties provided these compounds with a new route by way of the glucose transport carrier for intestinal absorption.

Pharmacokinetic evidence for the occurrence of extrahepatic conjugative metabolism of p-nitrophenol in rats.

p-Nitrophenol (PNP), as a model compound for the study of conjugative metabolism, was administered intravenously to rats. PNP and its conjugated metabolites, i.e. PNP-glucuronide (PNP-Glu) and PNP-sulfate (PNP-Sul), were determined in body fluids by reversed-phase high-performance liquid chromatography using ion-pair systems. Linear pharmacokinetics was applicable in the dose range of 1.6 to 8 mg/kg. The metabolic clearance which was obtained from the area under the PNP blood concentration curve (AUCiv) and from the excretion ratio of the total conjugates as PNP-Glu and PNP-Sul was so close to the hepatic blood flow that the PNP conjugation reactions seemed to be limited by the hepatic blood flow, that is the hepatic extraction ratio (EH) was expected to be 1. However, AUCpv, following portal vein administration of PNP (4 mg/kg), was not zero but was significantly different from AUCiv after the same dosing (P less than 0.05). Consequently, comparison between the AUC values from both dosing routes and the excretion ratio of PNP-Glu and PNP-Sul gave and EH of 0.43. Such a difference in EH obtained by the two methods suggested a contribution by extrahepatic conjugative metabolism. It was shown that the intrinsic hepatic clearance obtained, assuming exclusively hepatic conjugative metabolism, was certainly overestimated. Furthermore, the results of the conjugation reaction in tissue homogenates suggested a contribution by extrahepatic glucuronidation.

Heterogeneity of rat plasma albumin and drug binding.

Rat plasma albumin fractionated by Sephadex G-75 (superfine) gel filtration from freshly prepared rat plasma was further separated into three major fractions by chromatofocusing column chromatography. All the fractionated albumins had high binding affinity for the fluorescent probe 8-anilino-1-naphthalenesulfonic acid, as the model compound for acidic drugs, and were found to be immunologically identical.

Transport of sugars and amino acids in bacteria. XVI. Theory and evaluation of a model for the membrane transport reaction mediated by a single carrier with three binding sites for substrate.

A novel model is presented for bacterial active transport reactions which show a curvilinear Eadie-Hofstee plot and negative homotropic cooperativity in the kinetics of substrate uptake. Various models of a single carrier with multi-binding sites for substrate were constructed and examined theoretically. The fit of these models with experimental data on the kinetics of branched chain amino acid transport reactions were tested by iterative computation using the non-linear least square method. The transport model which fitted the experimental data best consisted of a single carrier with three binding sites for substrate in which one of the substrate-carrier complexes, CSS, is not active in translocating substrate across the cytoplasmic membrane. The mechanism of homeostatic regulation of the intracellular concentration of amino acids by active transport systems is discussed on the basis of this transport model.

Human genetically polymorphic deoxyribonuclease: purification, characterization, and multiplicity of urine deoxyribonuclease I.

A deoxyribonuclease I was purified from the urine of a 46-year-old male (a single individual) by using a series of column chromatographies to a homogeneous state as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was found to be a glycoprotein, containing 1 fucose, 7 galactose, 10 mannose, 6 glucosamine, and 2 sialic acid residues per molecule. The N-terminal amino acid sequence up to the 27th residue of the enzyme was similar to that of pancreatic deoxyribonuclease I from bovine and other species. The catalytic properties of the enzyme derived from a single individual closely resembled those of deoxyribonuclease I purified from human urine collected from several volunteers [Ito, K. et al. (1984) J. Biochem. 95, 1399-1406]. The purified enzyme was found to consist of multiple forms with different pI values. These findings are compatible with the existence of genetic polymorphism of deoxyribonuclease I in human urine previously reported [Kishi, K. et al. (1989) Hum. Genet. 81, 295-297]. This multiplicity of the urine enzyme might be due to variations in the primary structure and/or differences in the content of sialic acid.