Decreased expression of type II protein kinase C in HL-60 variant cells resistant to induction of cell differentiation by phorbol diester.

To evaluate the molecular basis for susceptibility of the cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), we examined biochemical activities and expression of subspecies of protein kinase C from HL-60 cells that are susceptible to differentiation induced by TPA and HL-60R cells, HL-60 variant cells that are resistant to such induction. Analysis of the subcellular distribution of protein kinase C revealed that the activity of this kinase in the cytosol from HL-60R cells was 30% of that from parental HL-60 cells but that the enzyme activities in the membrane showed similar values in these cells. Treatment of HL-60 cells with 100 nM TPA for 30 min resulted in a 75% decrease in protein kinase C activity in the cytosol and a 300% increase in this activity in the membrane. A minor subcellular redistribution of the enzyme activity was found in HL-60R cells after TPA treatment. Based on analysis of protein kinase C isozymes by hydroxyapatite column chromatography, the relative activities of types I, II, and III in the cytosol of HL-60 cells were 11, 80, and 9%, whereas those in HL-60R cells were 27, 36, and 37%, respectively. Type II isozyme was a major protein kinase C in the cytosol of HL-60 cells, but type II was less in the HL-60R cells. Among the three isozymes, type II enzyme was most sensitive to TPA with histone H1 as the substrate, although all three isozymes were activated Ca2+-dependently in the presence of phosphatidylserine. We suggest that the acquired resistance of HL-60R cells toward induction of cell differentiation by TPA may be associated with a decrease in the expression of the type II isozyme of protein kinase C.

Expression of three major protein kinase C isozymes in various types of human leukemic cells.

We examined the levels of protein kinase C (PKC) activity and the expressions of its three major isozymes, designated types I (gamma), II (beta), and III (alpha), in the cytosol and particulate fractions of cells from patients with acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL), in an attempt to elucidate the cell type- or lineage-specific expression of these isozymes. The levels of PKC activities in the cytosol and particulate fractions from AML cells were higher than those from ALL or CLL cells. The average PKC activities of AML cells, ALL cells, and CLL cells were 18.7, 12.2, and 11.3 pmol/min/10(8) cells, respectively, in the cytosol fractions and 4.4, 3.1, and 2.6 pmol/min/10(8) cells, respectively, in their particulate fractions. M1 cells (French-American-British classification) and AML cells with T-lymphocyte-associated surface antigens, such as CD2 and CD7, had significantly lower PKC activities among AML cells. Immunoblot analyses using monoclonal antibodies against each isozyme revealed that all three isozymes were broadly distributed on leukemic cells with considerable variability in the level of expression. All lymphoid leukemic cells expressed PKC-gamma in the cytosol fractions, albeit a minor component; however, this type was observed in cells from only half the number of AML patients. Those AML cells with cytosolic PKC-gamma usually expressed lymphoid surface antigens, such as CD2, CD7, and CD19. On the other hand, cytosolic PKC-beta and PKC-alpha were commonly observed in all types of leukemic cells. AML cells expressed these two types at almost equal levels, but in lymphoid cells, expressions of PKC-beta were usually more abundant than those of PKC-alpha. These data suggest that AML cells with lymphoid antigens might have a lower PKC activity but more predominant expression of cytosolic PKC-gamma than the usual AML cells.

Inhibition of platelet aggregation by the cAMP-phosphodiesterase inhibitor, cilostamide, may not be associated with activation of cAMP-dependent protein kinase.

We examined the involvement of cAMP-dependent protein kinase (A kinase)2 in the inhibition by cilostamide, a specific inhibitor of the low Km cAMP-phosphodiesterase (PDE), on 9,11-epithio-11,12-methanothromboxane A2 (STA2)-induced platelet aggregation. For comparative purposes, the PGE1 analogue, 17S-20-dimethyl-trans-delta 2-PGE1 (OP-1206) was used. OP-1206 (IC50 = 18 +/- 0.55 nM) and cilostamide (IC50 = 40 +/- 4.5 nM) were both potent inhibitors of the platelet aggregation induced by STA2 (1 microM). OP-1206 and cilostamide dose-dependently inhibited elevations in intracellular free Ca2+ ([Ca2+]i) caused by STA2. OP-1206 caused an almost complete inhibition of Ca2+ mobilization, but cilostamide did not prevent the STA2-induced elevation in [Ca2+]i to the same extent as OP-1206, even at a high concentration (greater than 200 nM). Cilostamide did not increase the cAMP level at concentrations (5-100 nm) which affected STA2-induced aggregation. OP-1206 significantly increased cAMP contents in platelets, and the degree of aggregation inhibition by OP-1206 appears to be related to the size of increase in cAMP. OP-1206 increased phosphorylation of the 50,000 mol. wt vasodilator-stimulated phosphoprotein, at concentrations of 7.9-79 nM, which inhibited aggregation induced by STA2. Cilostamide treatment resulted in a marginal increase in the 50,000 mol. wt phosphorylation at concentrations (10-100 nM) which completely inhibited the STA2-induced aggregation. (8R*, 9S*, 11S*)-(-)-9-Hydroxy-9-n-hexyloxy-8-methyl-2,3,9,10- tetrahydro-8,11-epoxy-1H, 8H, 11H-2, 7b, 11a-triazadibenzo(a,g)-cycloocta(c,d,e)trinden-1-one (KT-5720), a specific inhibitor of A kinase, not only reversed the inhibition by OP-1206 of STA2-induced platelet aggregation, but also inhibited the OP-1206-induced protein phosphorylation. However, the inhibition by cilostamide of STA2-induced aggregation was not prevented by pretreatment with KT-5720. Inhibition of the STA2-induced aggregation by OP-1206 may be associated with cAMP-dependent protein phosphorylation, while cilostamide may have inhibitory effects on STA2-induced platelet activation through mechanisms other than the activation of A kinase.

Genotyping of N-acetylation polymorphism and correlation with procainamide metabolism.

We studied the genotypes of polymorphic N-acetyltransferase (NAT2) in 145 Japanese subjects by the polymerase chain reaction-restriction fragment length polymorphism method. The rapid-type NAT2*4 was expressed at a higher frequency (68.6%) than the slow-type genes with specific point mutations (NAT2*6A, 19.3%; NAT2*7B, 9.7%; NAT2*5B, 2.4%). The frequency of NAT2* genotypes consisted of 44% of a homozygote of NAT2*4, 49% of a heterozygote of NAT2*4 and mutant genes, and 7% of a combination of mutant genes. The metabolic activity for procainamide to N-acetylprocainamide was measured in 11 healthy subjects whose genotype had been determined. Although the acetylation activity substantially varied interindividually, the variability was considerably reduced after classification according to the genotype. The N-acetylprocainamide/procainamide ratio in urinary excretion was 0.60 +/- 0.17 (mean +/- SD) for those with NAT2*4/*4, 0.37 +/- 0.06 for NAT2*4/*6A, 0.40 +/- 0.03 for NAT2*4/*7B, and 0.17 for NAT2*6A/*7B. The results indicated that the NAT2* genotype correlates with acetylation of procainamide.

CYP2C19 genotype-related efficacy of omeprazole for the treatment of infection caused by Helicobacter pylori.

OBJECTIVES: Omeprazole is used for the treatment of infection caused by Helicobacter pylori, and it is metabolized by the polymorphic cytochrome P4502C19 (CYP2C19). We have found that the anti-H pylori efficacy by the combination of omeprazole and antibiotics is related to the CYP2C19 genotype. METHODS: One hundred eight patients with cultured H pylori-positive gastritis or peptic ulcer were treated with three regimens: quadruple treatment without proton pump inhibitors (n = 25), dual treatment with omeprazole and amoxicillin (INN, amoxicilline) (n = 26), and triple treatment with omeprazole, amoxicillin, and clarithromycin (n = 57). The CYP2C19 genotype was determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and the assessment of the eradication of H pylori was based on all negative examinations, including culture, histology, and 13C-urea breath test. RESULTS: The eradication rates for the extensive metabolizers were 50% and 86% for the dual and triple treatments, respectively. In contrast, all of the poor metabolizers treated with omeprazole and antibiotics (n = 15) showed an eradication of H pylori. CONCLUSION: The anti-H pylori effect of dual treatment is highly efficient for CYP2C19 poor metabolizers, which suggests that clarithromycin is not necessary as a first line of therapy for this type of patients. Genotyping can provide a choice for the optimal regimen based on individual CYP2C19 genotype.

Alteration of intracellular actin levels induced by phorboldiester in human HL-60 leukemia cells susceptible or resistant to differentiation, and the effects of protein kinase inhibitors.

During monocyte-macrophage differentiation of HL-60 cells by 12-O-tetradecanoyl phorbol 13-acetate, the intracellular globular(G)-actin and polymerized(F)-actin increased 3-fold and 1.7-fold, respectively. Time course studies showed that these changes of actin levels were nearly coincident with the development of macrophage characteristics, including adhesiveness, positive reactivity against OKM-1 antibody and elevated lysozyme activity. When exposed to 5 nM TPA, these different properties of differentiation were detectable as early as 12 h after TPA treatment and reached a maximum by 24 h. Phosphorylation of 17 K and 27 K proteins, induced by TPA, occurred early (within 30 min) during TPA-induced differentiation. On the other hand, HL-60R cells, which are resistant to TPA in terms of the development of adhesiveness and differentiation, showed no change in both G- and F-actin levels, after the TPA treatment. TPA did not induce phosphorylation of these proteins in the HL-60R cells. In the presence of the protein kinase inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 20 microM) and staurosporine (10 nM), the increase in actin levels induced by TPA was inhibited as well as other later evidence of differentiation. These results suggest that the phosphorylation of specific proteins is closely associated with the process of differentiation of HL-60 cells into macrophages.

A role for protein kinase C in the growth of human erythroid progenitor cells.

We searched for a possible role for protein kinase C in the growth of human erythroid progenitor cells, using pharmacologic approaches. Two protein kinase C inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine, dose-dependently inhibited the growth of immature erythroid progenitor cells (BFU-E) induced by interleukin 3 (IL-3) plus erythropoietin (Ep) or granulocyte macrophage colony-stimulating factor (GM-CSF) plus Ep whereas a weaker analog of H-7, N-(2-guanidinoethyl)-5-isoquinoline sulfonamide (HA-1004), had no effect on the number of BFU-E. These three compounds had no effect on the growth of mature erythroid progenitor cells (CFU-E) stimulated by Ep. The culture of accessory cell-depleted bone marrow demonstrated that the effects of these compounds on colony formation do not appear to be mediated by accessory cells. The potential of these compounds to inhibit the GM-CSF-dependent growth of KG-1 cells correlated well with the extent of their inhibitor of protein kinase C activities from KG-1 cells. Thus, the protein kinase C system is apparently involved in the growth of BFU-E, supported by IL-3 or GM-CSF. The growth signal for CFU-E transduced by Ep may be achieved through other systems.

Gamma/delta T-cell lymphoma with hepatosplenomegaly: report of a case.

We describe the case of a patient with peripheral gamma/delta T-cell lymphoma (T-ML) with hepatosplenomegaly, generalized lymphadenopathy, and bone marrow involvement. A 44-year-old man had lymphoma, which became clinically apparent 2 months after the onset of myositis and insulin-dependent diabetes mellitus. A cervical lymph node biopsy specimen showed diffuse infiltration by large neoplastic cells with vascular proliferation. The neoplastic cells expressed the T-cell receptor (TCR)delta chain detected by TCR delta 1 and delta-TCS1, CD3, CD30, CD45RO, and epithelial membrane antigen, but not the TCR beta chain detected by beta F1, CD1a, CD2, CD4, CD5, CD7, CD8, CD25, HLA-DR, and terminal deoxynucleotidyl transferase. The cells had a clonal rearrangement of TCR gamma chain gene and a germ-line configuration of immunoglobulin heavy chain gene and TCR beta chain gene. Despite chemotherapy, the patient died of refractory lymphoma 4 months after diagnosis. Examination at autopsy revealed that the main hepatic and splenic neoplastic infiltration sites were the portal area and white pulp, respectively. Our patient differed from those with gamma/delta T-ML with hepatosplenic involvement reported previously with respect to the hepatic and splenic neoplastic infiltration patterns and the presence of lymphadenopathy.

Pharmaceutical approach to subcutaneous dosage forms of insulin.

The present studies were undertaken to describe the dynamic nature of the degradation and absorption of insulin in the subcutaneous injection site and to develop agents which would stabilize this dosage form. [125I]Insulin with 0.2-U/kg of unlabeled insulin in 10 microliters of aqueous solution was injected subcutaneously in rats under the depilated skin of the back. At various times, radioactive skin tissue was extracted and assayed for insulin and/or its metabolites by gel filtration. Using these data, absorption and degradation rate constants of these substances were estimated according to a one-compartment model. Absorption rate constants of insulin and its metabolite of low molecular weight (monoiodotyrosine) were 0.021 min-1 and 0.107 min-1, respectively, while the degradation rate constant for insulin to monoiodotyrosine was 0.013 min-1. Thus, the bioavailability of insulin injected subcutaneously was lower than expected, suggesting the necessity of stabilizing methods. The protection of insulin from degradation at the site of injection was examined by the addition of various peptides. It was found that benzyloxycarbonyl-Gly-Pro-Leu-Gly was a good stabilizing agent, and remarkably inhibited insulin degradation. This inhibition was confirmed by the increase of immunoreactive insulin level and the decrease of the blood glucose level. We postulated that this peptide protects the injected insulin from degradation by inhibiting the peptidase present in subcutaneous tissue.

Improved bioavailability of para-boronophenylalanine by cyclodextrin complexation.

This study was undertaken to develop an oral dosage form for para-boronophenylalanine (BPA) plus cyclodextrin (CD) for use in the thermal neutron capture therapy for malignant melanoma. Powders of the BPA and CD complexes were obtained in a molar ratio of 1:2. X-ray diffraction of the BPA-CD complexes showed halo patterns that indicated that each complex was in a new solid state as an amorphous compound. The enhancement of BPA solubility by glucosyl (G1)- and maltosyl (G2)-alpha-CD was greater than that with the other CDs. The isolation rate of BPA from its complex was different for each BPA-CD complex. The bioavailability of BPA in rats was improved with oral administration of the BPA-alpha-CD, G1-alpha-CD, and G2-alpha-CD complexes. In contrast, a complex of BPA and dimaltosyl (G2G2) or G2-beta-CD, which had low release rate and low solubility, did not improve the bioavailability of BPA. These results indicate that the solubility and release rate of BPA from a complex in solution are important for the bioavailability of BPA after oral administration of BPA-CD complexes.

Intratracheal delivery of peptide and protein agents: absorption from solution and dry powder by rat lung.

Proteins of high molecular weight and low lipophilicity must be administered parenterally to achieve the desired therapeutic blood levels. We investigated the absorption of peptide and protein agents by rat lung following their intratracheal administration, expressing it as percent bioavailability. An aqueous solution and/or a dry powder of calcitonin, insulin, thyrotropin stimulating hormone (TSH), follicle stimulating hormone (FSH), and human chorionic gonadotropin (HCG) was delivered into the exposed trachea of anesthetized rats, and blood was sampled from the jugular vein at specified intervals. The bioavailabilities of TSH, FSH, and HCG delivered in a solution of neutral pH were 2.5, 2.3, and 0.2 %, respectively. Transpulmonary absorption of a solution of these agents, administered with a surfactant or under acidic conditions, was 2-30 times greater than the values obtained in controls. The bioavailabilities of calcitonin, insulin, TSH, FSH, and HCG, given intratracheally as a dry powder, were 11.5, 6.5, 1.6, 0.6, and 0.1%, respectively. Following intratracheal administration, we noted a negative association between molecular weight and bioavailability. The intratracheal route may thus be useful for delivering peptide and protein agents.

Design of 9-beta-D-arabinofuranosyladenine (ara-A) transdermal delivery system for animal studies: regulation of drug concentration in vivo.

Transdermal delivery systems of 9-beta-D-arabinofuranosyladenine (ara-A), having controlling membranes of various permeabilities, were developed and applied to Azone-pretreated hairless mouse abdominal skin. It was confirmed that the blood concentrations of ara-A and its metabolite 9-beta-D-arabinofuranosylhypoxanthine (ara-H) in hairless mice are controlled by the permeability of the controlling membrane in the transdermal patch. Furthermore, these blood concentrations were found to closely agree with the values obtained from theoretical model calculations. Finally, but importantly, the "micropharmacokinetic" behavior of ara-A in cutaneous tissue could also be predicted. These results suggest that the transdermal patch may be employed in dermal and transdermal ara-A efficacy studies in the treatment of cutaneous herpes virus infections in hairless mice.

[Aplastic anemia which terminated in hypoplastic leukemia 10 years after diagnosis].

A 57-year-old woman was admitted to our hospital because of further examination of anemia in November, 1978. She was diagnosed as having aplastic anemia, which was not associated with any atypical findings. Treatment with oxymetholone had been effective. However, she developed pancytopenia in January, 1988. A bone marrow aspiration revealed a hypocellular marrow with 75.6% blasts. Following two courses with low dose cytosine arabinoside and vitamin D3, the leukemia improved although she needs red cell transfusions at intervals of one to two months because of persistent pancytopenia. We present a patient with aplastic anemia who developed a hypoplastic leukemia at the tenth year of the disease.

[Autopsy case of intravascular lymphomatosis with pancreatic carcinoma].

We report an autopsy case of intravascular lymphomatosis (IVL) that arose after radiation therapy and chemotherapy for an inoperable pancreatic carcinoma. A 66-year-old man who suffered from diabetes mellitus and pancreatic carcinoma presented with aggressive progression of consciousness disturbance and high fever. The laboratory findings disclosed marked thrombocytopenia, hypercalcemia, and elevated serum PTH-related peptide. The patient soon died of ventricular fibrillation due to uncontrollable hypercalcemia. Postmortem examination with immunohistochemical analysis revealed a well-differentiated tubular adenocarcinoma in the pancreatic body as well as an accumulation of neoplastic B-lymphocytes in small vessels throughout the body without systemic lymphadenopathy. To our knowledge, double neoplasms including IVL are extremely rare.

[A case of metastatic liposarcoma originating in the retroperitoneum successfully treated with combination chemotherapy].

We reported a 36-year-old woman with metastatic liposarcoma originating in the retroperitoneum, which responded well to adjuvant chemotherapy. The primary tumor was removed by surgery. Two months later, the patient developed metastasis to the brain, and to the lung four months later. Metastatic liposarcomas to the brain generally are extremely rare. The patient was treated with combination chemotherapy using cyclophosphamide, vincristine, adriamycin, and dacarbazine (CYVADIC). After she was examined, the former two drugs were alternated with vindesine and ifosfamide, and another regimen with cisplatin and etoposide was given after a three-week interval. As a result, both of the metastases totally disappeared. No recurrent lesion has been noted for two years. Although the role of chemotherapy for liposarcoma has not been well defined and little data support its use in an adjuvant setting, this combination chemotherapy seemed to be effective for advanced liposarcoma.

Fate of porcine and human insulin at the subcutaneous injection site. II. In vitro degradation of insulins in the subcutaneous tissue of the rat.

We compared the in vitro degradation of porcine and human insulin in the subcutaneous tissue of rat. The insulin degrading activity was largely confined to the 160000 X g supernatant fraction of subcutaneous tissue. The degradation of human insulin was approximately half that of porcine insulin in the supernatant fraction. The degradation of porcine insulin in subcutaneous tissue was inhibited by bacitracin, leupeptin, phosphoramidon, and Z-Gly-Pro-Leu-Gly, though the human insulin degradation was not. The degradation of both insulins was accelerated by glutathione. While the proteolytic enzyme activities of cathepsin-B and collagenase-like peptidase were detectable in subcutaneous tissue, chymotrypsin, elastase, kallikrein, alpha-thrombin, and trypsin activities were almost negligible. These in vitro studies suggest that human insulin is comparatively stable against proteolytic enzymes, probably collagenase-like peptidase or cathepsin-B, in the subcutaneous tissue, which support the in vivo evidence.

Fate of porcine and human insulin at the subcutaneous injection site. I. Degradation and absorption of insulins in the rat.

The plasma insulin and serum glucose levels were compared after the subcutaneous and intravenous administration of porcine and human insulin in rats. While no difference was observed in plasma insulin or serum glucose levels with either insulin after intravenous administration, the plasma insulin levels and hypoglycemic effects of human insulin were greater than those of porcine insulin after subcutaneous administration. At various time intervals, radioactivity in subcutaneous tissue was assayed for insulin and/or its metabolites by gel filtration. Using these data, the absorption and degradation rate constants of these substances were estimated according to a one-compartment model. The degradation rate constants of human insulin was approximately half of that for porcine insulin.

Clinical response to busramustine (KM-2210) in chronic lymphocytic leukemia: a pilot evaluation of estrogen receptor in relation to its therapeutic effect.

Busramustine (KM-2210), the benzoate of a 17 beta-estradiol-chlorambucil conjugate, was administered to 11 patients with chronic lymphocytic leukemia (CLL) which included eight cases of B-cell CLL and three cases of T-cell CLL. Four patients had received prior chemotherapy. Busramustine was given orally at an initial daily dose of 50-100 mg continuously, and the dose was modified according to hematological improvement. Two cases of B-cell CLL achieved clinical complete responses, six cases including two of T-cell CLL and four of B-cell CLL achieved partial responses and one case of B-cell CLL achieved improvement. The partial and complete response rate was 72.7%. Four patients showed estrogen receptor activity of CLL cells ranging from 3.5 to 57.5 fmol/mg cytosol protein, but there seemed to be no correlation between the estrogen receptor activity of the CLL cells and the therapeutic effects of busramustine. Toxic effects included diarrhea (2/11) and estrogen-related symptoms including breast pain (4/11), genital bleeding (2/5), gynecomastia (2/6) and loss of libido (2/6). The findings of this preliminary study suggest that busramustine is effective in the treatment of CLL, irrespective of the presence of the estrogen receptor.

The signal transduction systems and intracellular Ca2+ dynamics in arachidonate-induced platelet activation.

Types II and III protein kinase C were expressed in human platelets and showed slightly different modes of activation and kinetic properties. Type III isozyme was more sensitive than type II for the activation of each isozyme with arachidonate (AA) although both isozymes were activated by diacylglycerol and phosphatidylserine in a similar manner. When human platelets were stimulated by AA, two types of platelet activation, a low level of AA (0.1-2.5 micrograms/ml)- and a high level of AA (10-50 micrograms/ml)-induced activations, were observed. These activations were associated with the phosphorylation of 40K and 20K proteins. Although a low level of AA (0.45-10.0 micrograms/ml) induced the formation of [32P] phosphatidate in intact platelets prelabeled with [32P] Pi, AA at high concentrations (20-50 micrograms/ml) did not stimulate phospholipase C. The incubation of fura 2 loaded platelets with a low level of AA evoked a rapid and transient elevation in [Ca2+] i. In contrast, a high level of AA induced an irreversible increase in [Ca2+] i but this [Ca2+] i elevation alone was not associated with platelet activation. These results suggest that the signal transduction system by a high level of AA on human platelets is different from that seen with a low level of AA. A high level of AA induces platelet activation, without phospholipase C stimulation, and therefore, the ability of AA to directly activate protein kinase C (pre-dominantly type III isozyme) may contribute toward the activation of platelets by a high level of AA.

Cross-reactivity of TDX and OPUS immunoassay systems for serum digoxin determination.

The properties of the widely used TDX Analyzer and recently developed OPUS Immunoassay System were compared using 403 serum specimens taken from patients who did or did not take digoxin. Of the 210 specimens from patients not treated with digoxin, a false- positive digoxin concentration was detected in 15 specimens (7%) by TDX and in only 2 specimens (1%) by OPUS because of the cross-reactivity with structurally similar drugs. Potassium canrenoate, digitoxin, deslanoside, and methyldigoxin exhibited marked concentration-dependent cross-reactivity in the TDX assay method, whereas deslanoside and methyldigoxin only showed cross-reactivity with the antibody used in the OPUS method. Although a poor correlation was observed between these two methods for the determination of 193 samples from patients treated with digoxin, the correlation was remarkably improved (r = 0.914) and the slope approximated unity when excluded the data from patients who were treated concurrently with the cross-reactive compounds. In routine TDM of digoxin, the authors experienced two cases in which cross-reactivity of the assay system caused a clinical problem. Concurrent administration of intravenous canrenoate apparently interfered with the digoxin assay by TDX, but this problem was solved by using the OPUS system. The authors found OPUS more useful for monitoring serum digoxin concentrations in patients because of its superior specificity.