Significance of urinary glucaric acid measurement and its application to paclitaxel therapy.

Individual variations in P-450 activity affect the in vivo pharmacokinetics as well as the efficacy and side effect of drugs. It is proposed that urinary glucaric acid (GA) level may indirectly represent P-450 activity and may therefore be an indicator of P- 450 activity in the clinical setting. However, no standard method has been developed so far. Metabolism of paclitaxel (PTX), an anticancer drug, is mediated by P-450. If P-450 activity could be predicted by measuring urinary GA level during PTX administration and individual blood PTX concentration could be inferred, urinary GA level would be a potent tool to predict the efficacy and side effects of the drug. We therefore measured the urinary GA levels of patients on antiepileptics that are suggested to induce P-450 and those of control subjects, to determine whether urinary GA level could be an indicator of P-450 activity. Then, we examined the relationship between urinary GA level and blood PTX concentration and looked into the possibility of predicting pharmacokinetics based on the relationship between urinary GA level and area under the blood concentration-time curve (AUC). The means+/-S. D. of urinary [(GA level)/(Cr level) x 10] levels of 16 patients on antiepileptic medication and 24 control subjects were 0. 98 mg/mL+/-0. 91 and 0. 19 mg/mL+/-0. 07, respectively. The urinary GA levels of patients on antiepileptic medication were significantly higher than those of control subjects. On the other hand, the relationship between AUC and urinary GA levels in eight patients on PTX showed that AUC tended to become large when urinary GA levels were low. The above results reveal that measuring urinary GA level by the easy and noninvasive way of urine collection would enable us to predict P-450 activity and infer blood PTX concentration.

Clinical applications of urinary organic acids. Part I: Detoxification markers.

Modern instrumentation allows the measurement of organic acids in urine in their physiological concentration ranges. Eight of the compounds that are reported can serve as markers for specific toxicant exposure or detoxification challenges. Xylene exposure causes elevation of 2-methylhippurate, and orotic acid elevation reveals ammonia challenge that exceeds the capacity of the urea cycle. General hepatic detoxification stimulation by natural compounds, drugs, or xenobiotic compounds causes elevated levels of glucaric acid. Abnormalities of alpha-hydroxybutyrate, pyroglutamate, and sulfate can indicate up-regulated glutathione biosynthesis, impaired reformation of glutathione in the gamma-glutamyl cycle, and depleted total body glutathione status, respectively. Patterns of these compounds measured in a simple overnight urine specimen help to identify focal areas of clinical concern and monitor patient responses to detoxification interventions.

[Improvement of urinary d-glucaric acid assay and its application].

We propose an improved method to measure urinary D-glucaric acid (GA), which might be of value as an indirect index of the activity of cytochrome P450 (CYP). This method was about 20 times more sensitive than existing methods. Beer's law was obeyed in the concentration range 5.5-66 ng ml(-1) for GA, with the effective molar absorptivity at 533 nm and the relative standard deviation being 9.1 x 10(5) dm(3) mol(-1) cm(-1) and 0.69% (n = 6), respectively. In addition, we introduced the correction value {GA/Cr ratio x10} of urinary GA by measuring urinary creatinine (Cr) at the same time. Based on the proposed method, the GA and Cr values in spot urines of healthy persons and cancer patients were subsequently measured and the correction values of both groups subjected to comparison. As a result, a statistically significant difference was recognized between the two groups.

Biological monitoring screening of patients provided antineoplastic drugs including adriamycin, cyclophosphamide, 5-fluorouracil, methotrexate, and vincristine.

The aim of the present study was to establish screening biomarkers of exposure to antineoplastic drugs administered to 11 patients undergoing cancer chemotherapy. Among the anticancer drugs administered were cyclophosphamide (all), Adriamycin (5 of 11), methotrexate (3 of 11), 5-fluorouracil (4 of 11), vincristine (3 of 11), megestrol acetate (1 of 11), and procarbazine (1 of 11). The noninvasive urinary parameters investigated were thioethers, D-glucaric acid, elements, and forward and reverse mutagenesis using bacterial bioassays. The data were analyzed in terms of the observed concentrations and those corrected for personal baseline. Personal baseline correction for parameters with significant nonexposure baseline levels was essential. While glucaric acid and thioethers were increased by the drug treatments, the correlations with baseline-uncorrected data showing an inverse relationship proved spurious, because saturation of the detoxification systems occurred at the high doses administered. Glucaric acid was also influenced by methotrexate and vincristine. Thioether content was affected by cyclophosphamide only. The forward mutagenesis assay was directly correlated to cyclophosphamide dose but the reverse assay was not, in the presence or absence of rat S9 fraction. The forward assay was not sensitive to the effects of smoking. Relative to controls, the elements changed by cyclophosphamide were K, S, and P. Those affected by Adriamycin were Ca, Mg, and Na; 5-fluorouracil affected Ca, Mg, Na, and C; methotrexate changed P and S. The forward mutagenesis assay and D-glucaric acid concentrations were the screening biomarkers best suited to monitoring for extent of exposure to these antineoplastic drugs.

Paradoxical urinary excretion of D-glucaric acid in acute viral hepatitis.

The urinary excretion of D-glucaric acid and the plasma clearance of antipyrine were estimated during the acute phase of viral hepatitis and again during recovery. The plasma clearance of antipyrine was impaired during the acute stage of hepatitis, while the urinary excretion of D-glucaric acid was paradoxically high. Both parameters returned to normal during recovery. These findings suggest that the use of urinary D-glucaric acid excretion as an index of microsomal enzyme induction is unreliable when there is liver injury.

Urinary excretion of D-glucaric acid by severely burned patients.

The 24-hr excretion of urinary D-glucaric acid (UGA) has been measured in 5 seriously burned adults and compared with 6 healthy adults. In the burn patients mean UGA was 14.4 +/- 5.4 (+/- SD) mumoles/day and 28.7 +/- 6.5 mumoles/day in controls (p less than 0.002). In a 6-year-old female, UGA was also found to be very low. In a seventh burn patient, an adult male taking 20 mg of fluphenazine until his injury, his UGA was still in the normal range (29 mumoles/day) on the day of admission but descended to 21 mumoles/day at 2 days, to 16 at 4 days, and to 13 at 6 days. Treatment with fluphenazine was then reinstituted and on the tenth day UGA was 28 mumoles/day, indicating that after thermal injury UGA can respond to drugs. Although the inference has not been proved that decreased UGA corresponds to a decreased activity of drug metabolism, there is evidence of a strong correlation between increased UGA and increased drug metabolism. A decrease of UGA in disorders that generally lower metabolic activity supports a possible correlation in severe burns. If drug metabolism activity is lowered in the seriously burned patient, drug overdose may well result from the usual clinical doses.

Time-course of interaction between carbamazepine and clonazepam in normal man.

The applicability of a pharmacokinetic model for drug interactions by enzyme induction was tested by chronic dosing situation using carbamazepine (Tegretol) as the inducer and clonazepam (Clonopin) as the drug affected. Seven healthy subjects received one 1.0 mg clonazepam tablet once a day for 29 days and one 200 mg carbamazepine tablet once a day from days 8 to 29. Plasma levels of clonazepam were measured by electron-capture gas-liquid chromatography and those of carbamazepine and its epoxide metabolite by gas chromatographic-chemical ionization-mass spectrometry. Clonazepam plasma levels reached an initial steady-state by day 7 and declined to a lower steady-state over 5 to 15 days after additions of carbamazepine. The decrease in clonazepam levels ranged between 19% and 37%. Autoinduction of carbamazepine metabolism was also evident. Urinary excretion of D-glucaric acid increased 2- to 4-fold following carbamazepine administration (p less than 0.005). This increase provided additional evidence that the present interaction was due to enzyme induction. Experimental clonazepam levels were fitted to an induction pharmacokinetic model for multiple dosing with an exponentially increasing clearance. Induced half-lives of clonazepam (mean = 22.5 +/- 11.5 hr) were shorter (p less than 0.005) than control values (32.1 +/- 16.6 hr). Apparent enzyme(s) turnover half-lives ranged between 1 and 6 days.

Rapid normalization of antipyrine oxidation by heme in variegate porphyria.

Effects of heme on hepatic xenobiotic drug metabolism were investigated in eight subjects with variegate porphyria. A single infusion of heme arginate (3 mg/kg heme) reversed rapidly the prolonged mean elimination half-life of antipyrine from 27.2 to 12.7 hours (p less than 0.001) and increased total clearance from 0.23 to 0.44 ml/min/kg (p less than 0.001). Excretion of 6 beta-hydroxycortisol and D-glucaric acid increased significantly during heme infusion. Excretion of urinary porphyrin precursors increased during the antipyrine test but was normalized by heme. It is concluded that in variegate porphyria a partial block in heme biosynthesis results in subnormal capacity of P450-associated monooxygenases, but this is easily normalized by exogenous heme.

Effects of felbamate on the pharmacokinetics of phenobarbital.

The effects of felbamate on the pharmacokinetics of phenobarbital and one of its main metabolites, parahydroxyphenobarbital, were assessed in a parallel-group, placebo-controlled, double-blind study, in 24 healthy volunteers. Pharmacokinetic parameters of phenobarbital and parahydroxyphenobarbital were determined from plasma and urine samples obtained after 28 days of daily administration of 100 mg phenobarbital and after a further 9 days of phenobarbital plus 2400 mg/day felbamate or placebo. Felbamate increased phenobarbital values for area under the plasma concentration-time curve from 0 to 24 hours and maximum concentration by 22% and 24%, respectively, whereas placebo had no effect. This increase was caused by a reduction in parahydroxylation of phenobarbital and possibly through effects on other metabolic pathways. Because felbamate inhibits the S-mephenytoin hydroxylase (CYP2C19) isozyme in vitro, it appears that phenobarbital hydroxylation is mediated in part by this isozyme.

Urinary D-glucaric acid excretion during rifampicin/isoniazid and anticonvulsant enzyme induction.

As measured by urinary D-glucaric acid excretion, an index of hepatic enzyme induction, glutethimide was the most powerful of six such inducers tested. In patients with tuberculosis, rifampicin, 450 mg daily, induced excretion rates of the lower dose range of anticonvulsants in epileptics. The effect was detectable in the first few days but the degree and rate of rise to maximum excretion were variable. This may be due either to disposition of rifampicin or to genetic susceptibility to enzyme induction. Plasma beta-glucuronidase, an essential enzyme of the glucuronic acid pathway, could be induced independently of an increase in D-glucaric acid excretion. Plasma gamma-glutamyltranspeptidase-levels, an index of hepatic microsomal enzyme induction, were elevated in only 20 of 83 subjects receiving rifampicin and isoniazid, and in all of them urinary D-glucaric acid excretion was normal. Neither of these indices, therefore, showed hepatic enzyme induction during combined therapy when other pathways such as oxidative metabolism continued to be induced. Different active sites of rifampicin and isoniazid on glucuronic acid and other biochemical pathways emphasize the complexity of final metabolic effects in patients on long-term therapy.

Hepatic enzyme induction patterns and phenothiazine side effects.

D-Glucaric acid excretion was followed in psychotic patients treated with phenothiazines for 12 days and in a control group of subjects who had no psychiatric disease. About half the psychiatric patients had a treatment-related rise in D-glucaric acid excretion compatible with enzyme induction. These patients had fewer and less severe neurologic side effects than those who did not have a significant rise in urinary D-glucaric acid levels. It is concluded that individual differences in metabolism of phenothiazines may in part account for the variability in clinical response to these drugs.

Antipyrine pharmacokinetics and D-glucaric excretion in kwashiorkor.

Hepatic microsomal oxidation and glucuronidation were studied in 15 children with kwashiorkor on admission to the hospital and again after 3 weeks of nutritional rehabilitation. Microsomal oxidation as measured by antipyrine half-life and clearance was shown to be depressed in the acute phase of malnutrition (T 1/2 = 7.9 +/- 5.0 hr, clearance = 8.4 +/- 5.1 ml/min) improving with nutritional rehabilitation (T 1/2 = 4.3 +/- 2.3 hr, clearance = 15.5 +/- 8.7 ml/min). Urinary D-glucaric acid excretion increased from 60.6 +/- 42.2 mumoles/24 hr to 121.8 +/- 105.0 mumoles/24 hr over the same time period. Evidence is thus presented that both hepatic microsomal oxidation and glucuronidation are depressed in the acute phase of kwashiorkor but recover with nutritional rehabilitation.

Studies on the biochemical effects of the aldose reductase inhibitor 2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dione (Al 1576, HOE 843). Detection of D-glucaric and D-glucuronic acid excretion by high resolution 1H and 13C NMR spectroscopy.

The effects of two aldose reductase inhibitors on the biochemical composition of rat urine were investigated using high resolution 1H and 13C NMR spectroscopy. We report the elevated excretion of D-glucaric acid (DGA) and D-glucuronic acid (GCA) following treatment with 2,7-difluorospirofluorene-9,5'-imidazolidine-2'4'-dione (Imirestat, IM, Al 1576, HOE 843) at 50 mg/kg/day for 1 month, but not with 3-4-bromo-2-fluorobenzyl-4-oxo-3-phthalazine-1-ylacetic acid (Ponalrestat, Statil), dosed at 50 mg/kg/day for 2 weeks. Sugar aciduria was also detected following treatment with the cytochrome P450 inducer phenobarbitone (PB) at 45 mg/kg/day for 1 month, although the qualitative and quantitative pattern of excretion of sugar acids differed greatly between the IM and PB treatment groups. The levels of GCA excreted are elevated 11-fold by IM treatment from 19.0 to 210.0 mumol/24 hr, but only 2.5-fold by PB, from 9.7 to 23.9 mumol/24 hr. DGA was not detectable in control urine, although levels did increase by 30% during the study from 7.5 to 10.9 mumol/24 hr, between day 8 and day 29, with IM treatment, and by 60% from 1.7 to 4.9 mumol/24 hr following PB administration for the same time period. This predominant elevation of DGA and GCA caused by IM treatment far exceeds previous records. In contrast, PB treatment resulted in an increase in intensity of a number of partially resolved sugar resonances, but at a much lower level than resulted from IM treatment. A raised level of DGA and GCA is usually associated with hepatic P450 induction; however, we report here profound DGA and GCA uria as a result of the inhibition of the aldehyde reductase, hexonate dehydrogenase (EC 1.1.1.19, EC 1.1.1.20). This mechanism is not closely linked to P450 induction, corroborating the current view that elevated excretion of DGA is not a reliable indicator of hepatic enzyme induction. This study further demonstrates the use of high resolution NMR spectroscopy in the detection of a novel biochemical effect which may go unnoticed during routine clinical chemistry tests.

Biological monitoring of workers exposed to N-nitrosodiethanolamine in the metal industry.

Biological monitoring of occupational hazards was performed in workers using cutting fluids containing N-nitrosodiethanolamine (NDELA). The study involved a group of 25 male subjects from some metal factories in central Italy who used cutting fluids with an NDELA content of > or = 5 mg/l (high-exposure group) and a group of 37 males exposed to cutting fluids with an NDELA content < 5 mg/l (low-exposure group). For comparison, we recruited a control group consisting of 37 subjects living in the same area. For all subjects, internal dose (urinary excretion of NDELA, mutagens, and thioethers), early biological effects (sister chromatid exchanges in blood peripheral lymphocytes), and urinary excretion of D-glucaric acid (DGA) as an endpoint product in the glucuronidation pathway were assessed. The results showed that only the workers using cutting fluids with NDELA concentrations of > or = 5 mg/l excreted trace amounts of NDELA in their urine. Urine excretion of mutagens was similar in the two exposure groups and in the controls. High-exposure subjects had a higher mean value of urinary thioethers than low-exposure and control subjects, but no differences were found in urinary DGA or lymphocyte sister chromatid exchange among the three groups. Smoking status increased the mean values of all the biomarkers, and coffee drinking was associated with urinary DGA excretion.

Biological monitoring of genotoxic hazard in workers of the rubber industry.

Biological monitoring of genotoxic hazard in the rubber industry was performed in 19 male workers and 20 age-matched controls in a local health unit in northern Italy. Peripheral blood lymphocytes were analyzed for the presence of DNA damage (single-cell microgel-electrophoresis, or comet assay) and for cytogenetic parameters (sister chromatid exchanges and micronuclei frequency, and proliferative rate index). The following bioassays were performed in urine samples: a) mutagenicity test and concentration of thioethers as markers of exposure, and b) excretion of D-glucaric acid and 6-beta-hydroxycortisol (related to 17-hydroxycorticosteroid excretion) as indicators of the inductive status of the microsomal enzyme system (phase-I). The exposed subjects showed statistically higher mean values of 17-hydroxycorticosteroids and micronuclei and lower values of 6-beta-hydroxycortisol than controls, when taking cigarette smoking into account. The comet assay showed higher values for migration distance in exposed subjects than controls, although the differences were not significant at a p-value of 0.05. These findings suggest that industrial exposure in the rubber processing industry may cause genetic damage and may modify the activity level of some enzymes; these results should be considered with caution due to the small number of subjects enrolled.

Possible induction of cholinesterase in epileptic patients treated with anticonvulsant drugs: relationship with lipoprotein levels.

The effect of enzyme-inducing anticonvulsant drugs on the serum concentrations of lipoproteins has been widely studied. However, there is little agreement between the results with regard to the possible development of a lipoprotein profile related to an increased or decreased cardiovascular risk. It has been suggested that cholinesterase (ChE) could be induced by these drugs, something of undeniable interest as ChE appears to have a relation to the metabolism of lipoproteins. The serum activity of ChE was determined in a group of 90 adult epileptic patients (56 male and 34 female) treated with phenobarbital, phenytoin, and carbamazepine. The liver enzyme induction produced by these drugs was then evaluated by determining serum gamma-glutamyltranspherase activity and urinary excretion of D-glucaric acid. A significant increase of serum ChE (p < 0.005) was found in the group of patients compared to a control group (n = 49) with a similar distribution for age and sex. A significant correlation was found for both male and female patients between ChE and concentrations of triglycerides, phospholipids, cholesterol, low-density lipoprotein (LDL) phospholipids, LDL-cholesterol, and apolipoprotein B (p < 0.01). Similarly, in female patients, ChE had a significant correlation with the total cholesterol/high-density lipoprotein (HDL) cholesterol and LDL-cholesterol/HDL-cholesterol ratios (p < 0.01). The ChE/HDL-cholesterol relationship, which has been proposed as a marker for cardiovascular risk, presented significant correlations with the total cholesterol/HDL-cholesterol and LDL-cholesterol/HDL-cholesterol ratios in patients of both sexes (p < 0.001). In the case of epileptic patients treated with enzyme-inducing anticonvulsant drugs, there may be an association between the possible induction of ChE and the metabolism of lipoproteins containing apolipoprotein B.

Urinary D-glucaric acid and serum hepatic enzyme levels in chronic alcoholics.

Urinary D-glucaric acid (DGA) and the activities of gamma-glutamyl transferase (GGT) and other hepatic enzymes in serum were determined in 33 noncirrhotic male alcoholics who had continued to consume alcohol until at least 24 h prior to the taking of samples. DGA excretion was significantly greater in them than in a group of 30 healthy controls (p less than 0.001), exceeding the upper reference level in 38% of the alcoholic cases (as compared with 88% for GGT). In the alcoholic patients, there was highly significant correlation between urinary DGA and serum GGT (r = 0.613, p less than 0.001), suggesting that in both cases the increased levels are due to enzyme induction. None of the biochemical variables studied were significantly correlated with estimated daily alcohol consumption. Urinary DGA levels fell off rapidly with abstinence, and in 31 alcoholic patients who had consumed no alcohol for 5 days, there was no statistically significant correlation between DGA excretion and serum GGT (r = 0.158, p congruent to 0.4).

Urine mutagenicity and biochemical parameters as markers of exposure to petroleum pitch using a rat model.

A petroleum pitch sample collected in a carbon electrode factory was studied using a series of in vivo assays for genotoxicity and enzymatic induction capability. Rats were treated with the petroleum derivative in three doses: 100, 50, and 10 mg/kg body weight. The treatment produced a rapid excretion of mutagenic substances in the urines of the first 24 hr only in rats treated with high doses (100 and 50 mg/kg). No faecal mutagenic activity was observed. Analyses of urinary thioethers showed that urinary metabolites derived from the compounds present in the pitch-sample at the lowest dose-administered (10 mg/kg) were eliminated primarily as cysteine conjugates. The pitch sample was found to be a good inducer of pulmonary and hepatic aryl hydrocarbon hydroxylase, especially after a 50 mg/kg dose. Urinary D-glucaric acid content was always statistically increased in treated animals compared with controls, confirming the enzymatic induction activity. Hepatic glutathione-S-transferase activity increased following treatment with 50 and 10 mg/kg doses.

Influence of sex, age, and smoking habits on the urinary excretion of D-glucaric acid.

Reference values for urinary D-glucaric acid and the influence of sex, age and smoking habits were evaluated with a low-pH enzymatic method. D-Glucaric acid measured on spot urine samples from 573 healthy subjects gave mean concentrations (mumol/1) and D-glucaric acid/creatinine ratios (mmol/mol creatinine) of 56.1 (+/- 22.9) and 3.05 (+/- 0.99) for males and 53.3 (+/- 20.9) and 3.35 (+/- 0.95) for females. No difference between morning and evening was observed for urinary D-glucaric acid/1 values, but D-glucaric acid/creatinine was higher in the evening samples for both sexes. There was a negative correlation between D-glucaric acid/1 values and age in males but not in females: the decrease of D-glucaric acid concentration was, however, quantitatively very small. Smoking produced a significant increase in D-glucaric acid concentration and in the D-glucaric acid/creatinine ratio for males and also partially for females.

Low-pH method for the enzymatic assay of D-glucaric acid in urine.

The enzymatic methods for measuring D-glucaric acid in urine are based on the conversion of D-glucaric acid into its 1,4-lactone and measurement of inhibition of 1,4-lactone against beta-glucuronidase at pH 5.0. All the enzymatic methods described suffer from the disadvantage of a procedure that is complicated and inherently inaccurate, because the nature of glucaric acid/1,4-lactone equilibrium has not been properly considered in the development of such methods. After elucidating the factors influencing glucaric acid/1,4 lactone equilibrium in more detail, a low-pH enzymatic method has been developed in which the 1,4-lactone is formed in the urine sample by acid boiling at pH 3.8 and assayed at the same pH using beta-glucuronidase from Limpets. This procedure allows the acid/lactone equilibrium to remain stable during both the lactonization step and the enzymatic assay. The coefficient of variation for the proposed method (within-run and between-day precision) was from 4.2 to 8.7. The analytical recovery varied from 92-108%.