Speciation analysis of arsenic in urine samples from APL patients treated with single agent As2O3 by HPLC-HG-AFS.

Arsenic trioxide [As2O3, arsenite (AsIII) in solution] has been applied successfully for the treatment of acute promyelocytic leukemia (APL). The arsenic speciation analysis of urine is critical to reveal the metabolic mechanism and the relationship between arsenic species and the clinical response. To characterize the arsenic species in urine, a simple and robust HPLC-HG-AFS method was developed and validated to quantify the levels of arsenic species [AsIII and its metabolites, monomethylarsonic acid (MMAV), dimethylarsinic acid (DMAV), and arsenate (AsV)] in urine samples from 66 patients with APL. Patients received As2O3 (0.16 mg/kg/day) via continuous slow-rate infusion or conventional infusion. Urine samples were collected at steady state before the start of the next daily administration. The relative proportions (median) of arsenic species in urine were: AsIII, 33.00% (IQR: 24.34%-46.82%); DMAV, 36.42% (IQR: 25.82%-51.98%); MMAV, 23.89% (IQR: 19.52%-27.19%); and AsV, 2.22% (IQR: 1.293%-3.665%). The levels and proportions of arsenic species vary widely among individual patients. DMAV and un-metabolized AsIII were the dominant arsenic compounds excreted from the urine of patients with APL treated with As2O3. AsV was the least abundant arsenic species in all urine samples. Good positive correlations were found between the levels and proportions of arsenic species in urine and those in plasma; thus, urinary arsenic can reflect the levels of arsenic in plasma. Urinary arsenic is a critical biomarker to evaluate the metabolism and toxicity of arsenic in the clinical application of As2O3.

AS3MT Polymorphisms, Arsenic Metabolism, and the Hematological and Biochemical Values in APL Patients Treated with Arsenic Trioxide.

Although arsenic has shown remarkable therapeutic efficacy in patients with acute promyelocytic leukemia (APL), its side effects are rarely reported. In this article, the associations among urinary arsenic profiles, hematological and biochemical values, and 3 AS3MT genotypes (rs3740392, rs3740390, and rs11191439) were evaluated in 50 APL patients treated with arsenic trioxide (As2O3). Results revealed the levels of serum enzymes (ALT, AST, and GGT), GLU and the count of WBC and NEUT#, which were markers of hepatic damage, diabetes and leukocytosis, respectively, were increased significantly 10 days after the administration of As2O3. The percentages of dimethylated arsenic (DMA) and the secondary methylation index (SMI, DMA/MMA) were negatively associated with the levels of ALT and AST. Patients with the AS3MT rs3740390 TC or TT genotype, compared with rs3740390 CC genotype, had significantly higher levels of DMA%, SMI and significantly lower levels of ALT and AST. Furthermore, the frequency for the heterozygous variant of rs11191439 was absolute low (N = 1). For rs3740392, no statistical differences were noted in urinary arsenic profiles and hematological and biochemical values in individuals with different genotypes. These results indicate that inherent genetic information of the AS3MT rs3740390 genotypes is a novel predicted or evaluated target for As2O3-induced side effects and therapeutic efficacy for the treatment of APL.

Exceptions in patterns of arsenic compounds in urine of acute promyelocytic leukaemia patients treated with As2O3.

Arsenic trioxide (As(III) in solution) has been shown to be the most active single agent in combating acute promyelocytic leukemia (APL). It is metabolized and excreted via urine as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and As(V), along with excess As(III). In our study eight APL patients were treated (intravenously) with 0.15 mg As2O3/kg/day. During the therapy As(III) and its metabolites were followed in pre- and post-infusion urine using HPLC for separation followed by on-line detection using hydride generation-atomic fluorescence spectrometry. Five patients had a normal excretion pattern of residual arsenic compounds in morning pre-infusion urine, with 15-25% of As(III), 35-55% of DMA, 25-30% of MMA and 1-5% of As(V), while three patients showed unexpected exceptions from typical excretion patterns of arsenic compounds (i) a high DMA/MMA ratio (factor 5.3), (ii) severe As(III) oxidation (10.2% As(III) converted to As(V)) or (iii) the presence of an excessive amount of As(III) (average 30.4% of total arsenic). Intriguing was the occurrence of post-infusion oxidation of As(III) to As(V) observed in almost all patients and being especially high (>40%) in patient with increased residual As(V). Results indicate that arsenic metabolites patterns can be unpredictable. Observed high levels of un-metabolised As(III) are a warning signal for side effects and for routine determination of arsenic metabolites during first days of treatment. High or low percentages of MMA or DMA did not show any observable effect on treatment results, while clear presence of post-infusion As(V) supports theoretical claims of in vivo oxidation (detoxification) of As(III) to As(V) associated with various metabolic processes.

Arsenic methylation metabolism and liver injury of acute promyelocytic leukemia patients undergoing arsenic trioxide treatment.

Although arsenic is effective in the treatment of acute promyelocytic leukemia (APL), as a well-known environmental toxicant, the side effects of arsenic treatment and arsenic methylation metabolism of the patients are rarely reported. In this manuscript, we investigated 23 APL patients treated with 10 mg arsenic trioxide daily, detected the arsenic metabolites in urine samples collected on the 0, 10th, and 20th day of arsenic treatment. At the same time, liver function and blood routine examination were also investigated in these APL patients. We found that, urinary monomethylated arsenic proportion (MMA%) increased, but dimethylated arsenic proportion (DMA%), the first methylation ratio (FMR) and the secondary methylation ratio (SMR) decreased with consecutive administration of arsenic trioxide. After adjustment for age impact, no statistical difference was observed in urinary arsenic concentrations and arsenic methylation capacity between male and female at the same treatment time point. During arsenic trioxide treatment of APL, transient elevation of serum aminotransferases was found in the blood samples, which indicated that liver injury occurred and probably reversible. Leukocytosis developed in 5 of the 23 patients with the administration of arsenic trioxide. No statistical difference was observed in the other blood routine examination parameters. These results demonstrated that the capacity of arsenic methylation decreased and transient liver injury occurred during arsenic trioxide treatment of APL, which indicated that the side effects of clinical arsenic treatment can not be ignored.

Analytical artefacts in the speciation of arsenic in clinical samples.

Urine and blood samples of cancer patients, treated with high doses of arsenic trioxide were analysed for arsenic species using HPLC-HGAFS and, in some cases, HPLC-ICPMS. Total arsenic was determined with either flow injection-HGAFS in urine or radiochemical neutron activation analysis in blood fractions (in serum/plasma, blood cells). The total arsenic concentrations (during prolonged, daily/weekly arsenic trioxide therapy) were in the microg mL(-1) range for urine and in the ng g(-1) range for blood fractions. The main arsenic species found in urine were As(III), MA and DMA and in blood As(V), MA and DMA. With proper sample preparation and storage of urine (no preservation agents/storage in liquid nitrogen) no analytical artefacts were observed and absence of significant amounts of alleged trivalent metabolites was proven. On the contrary, in blood samples a certain amount of arsenic can get lost in the speciation procedure what was especially noticeable for the blood cells although also plasma/serum gave rise to some disappearance of arsenic. The latter losses may be attributed to precipitation of As(III)-containing proteins/peptides during the methanol/water extraction procedure whereas the former losses were due to loss of specific As(III)-complexing proteins/peptides (e.g. cysteine, metallothionein, reduced GSH, ferritin) on the column (Hamilton PRP-X100) during the separation procedure. Contemporary analytical protocols are not able to completely avoid artefacts due to losses from the sampling to the detection stage so that it is recommended to be careful with the explanation of results, particularly regarding metabolic and pharmacokinetic interpretations, and always aim to compare the sum of species with the total arsenic concentration determined independently.

Arsenic speciation in urine from acute promyelocytic leukemia patients undergoing arsenic trioxide treatment.

Arsenic has been used successfully in clinical trials for treating acute promyelocytic leukemia (APL). Although sublethal doses of inorganic arsenic are used, little is known about the pharmacokinetics and metabolism of the high levels of arsenic in APL patients. To fill this important gap, this study describes the speciation of arsenic in urine from four APL patients treated with arsenic. Each patient was injected daily with an arsenite (As(III)) solution that contained 10 mg of As(2)O(3) precursor. Speciation analysis of the patient urine samples collected consecutively for 48 h, encompassing two intravenous injections of arsenic, revealed the presence of monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), monomethylarsonic acid (MMA(V)), and dimethylarsinic acid (DMA(V)). The intermediate methyl arsenic metabolites, MMA(III) and DMA(III), were detected in most urine samples from all of the patients when a preservative, diethyldithiocarbomate, was added to the urine samples to stabilize these trivalent arsenic species. The major arsenic species detected in the urine samples from the patients were As(III), MMA(V), and DMA(V), accounting for >95% of the total arsenic excreted. The relative proportions of As(III), As(V), MMA(V), and DMA(V) in urine samples collected 24 h after the injections of As(III) were 27.6 +/- 6.1, 2.8 +/- 2.0, 22.8 +/- 8.1, and 43.7 +/- 13.3%, respectively. The relatively lower fraction of the methylated arsenic species in these APL patients under arsenic treatment as compared with that from the general population exposed to much lower levels of arsenic suggests that the high levels of As(III) inhibit the methylation of arsenic (inhibits the formation of methyl arsenic metabolites). The arsenic species excreted into the urine accounted for 32-65% of the total arsenic injected. These results suggest that other pathways of excretion, such as through the bile, may play an important role in eliminating (removing) arsenic from the human body when challenged by high levels of As(III).

[Pharmacokinetics of intravenous arsenic trioxide in the treatment of acute promyelocytic leukemia].

OBJECTIVE: In order to study the pharmacokinetics and metabolism of arsenic trioxide (As22O3), and its major side effects. METHODS: The clinical pharmacokinetics of intravenous As2O3 in 8 relapsed acute promyelocytic leukemia (APL) patients were studied by Gas-phase chromotography. RESULTS: (1) After 2 hour intravenous drips of 10mg As2O3, drug plasma maximal concentration (Cpmax) was 0.94 +/- 0.37mg/L(x +/- s), time to peak concentration (Tp) was 4hrs, T1/2 alpha and T1/2beta were 0.89 +/- 0.29hrs and 12.13 +/- 3.31hrs, respectively, apparent volume of distribution (Vc) was 3.83 +/- 0.45L, system clearance (CLs) was 1.43 +/- 0.17L/h and area under curve (AUC) was 7.25 +/- 0.97mg x h/L. Importantly, the continuous administration of As2O3 did not alter the pharmacokinetic behaviors. (2) During As2O3 therapy, 24-hour arsenic content in urine accounted for 1%-8% of the total dose (10mg) per day. And it decreased gradually after drug withdrawal. (3) Arsenic contents in nail and hair were increased continuously during As2O3 therapy. The peak concentrations could be five to seven-fold higher than that pre-treatment. It also decreased gradually after drug with drawal. CONCLUSION: As2O3 is a relatively safe and effective drug in the treatment of APL patients, in spite of certain accumulation in some tissues.

Epidermal growth factor in acute promyelocytic leukemia treated with retinoic acid.

We studied 18 patients with acute promyelocytic leukaemia and 13 with relapsed APL. We found a significantly elevated EGF in acute leukaemia, especially in APL, being 418.59 +/- 19.2 micrograms in the 24-h urine that was much higher than that of the normal controls. When eight APL patients achieved complete remission by RA treatment, the EGF value decreased to 149.9 +/- 27.3 micrograms in the 24-h urine near to normal. In 13 patients with relapsed APL, EGF rose to 446.9 +/- 82.6 micrograms in the 24-h urine. Most interestingly, this elevated EGF could be detected before the relapse by 5 +/- 0.84 months in seven out of eight APL with relapse. We suggest that the unaccountably elevated EGF during remission period may be an indicator of the occurrence of relapse.