Kinetics of chlorambucil in vitro: effects of fluid matrix, human gastric juice, plasma proteins and red cells.

The mechanisms involved in the bioavailability of chlorambucil or 4-[p-(bis[2-hydroxyethyl]amino)phenyl]-butyric acid are poorly understood. The effects of different matrices on the disintegration of chlorambucil were investigated by HPLC, 1H NMR, 31P NMR, and mass spectrometry. Cellular incorporation and protein binding of the drug in vitro was assessed with [3H]-chlorambucil. Decomposition of chlorambucil and its major metabolite, phenylacetic acid mustard, to mono- and dihydroxy derivatives, was significantly faster in water than in PBS, (phosphate-buffered saline, pH 7.4). The hydrolysis of chlorambucil was as fast in plasma ultrafiltrate as in PBS; plasma proteins, preferentially albumin, prevented this disintegration. In phosphate-buffered media, two additional stabile hydrolysis products were found which were characterised as the mono- and bis-phosphates of 4-[p-(bis[2-hydroxyethyl]amino)phenyl]butyric acid, results of the reaction of nucleophilic buffer species with the aziridinium ion intermediates. Chlorambucil bound covalently to plasma proteins and was incorporated into red cells. These interactions are likely to have a significant role in vivo, reducing the bioavailability of the drug. High H+ concentration associated with high chloride concentration in human gastric juice had a stabilizing effect on chlorambucil. Incorporation of [3H]-chlorambucil into red cells was inhibited in a concentration-dependent fashion by whole human plasma as well as by albumin. We conclude that the chemico-biological interactions demonstrated in the present investigation provide explanations for the remarkable pharmacokinetic differences observed intra- and inter-individually in the clinical use of chlorambucil. The present information is important, when clinical or in vitro evaluation of efficacy and bioavailability of chlorambucil is considered.

The effect of ethanol or hepatotoxin exposure on rat transferrin desialylation.

Serum carbohydrate-deficient transferrin (CDT) is being increasingly used as a biological indicator for excessive alcohol consumption. However, the mechanisms behind the changes in the carbohydrate moiety of transferrin are unclear, although they have been suggested to be mediated by acetaldehyde or liver damage. To study this, an animal model involving alterations in serum isotransferrin concentrations would be needed. The present work examined the changes in the carbohydrate moiety of transferrin in rats after different degrees of ethanol exposure, the effects of chronically elevated acetaldehyde levels, and also the changes, produced with liver toxins (galactosamine) and carbon tetrachloride). Ethanol was administered both in the drinking fluid and by intubation, reaching a dose of 11 g/kg/day over 7 weeks, or 16 g/kg/day over 4 weeks. Serum samples from rats maintained on high ethanol for 10 weeks by intragastric infusion were also analysed. Some rats simultaneously had cyanamide administered to elevate acetaldehyde levels. However, neither ethanol nor acetaldehyde had any effect on transferrin. Intraperitoneal galactosamine, but not carbon tetrachloride, induced transferrin desialylation. Thus, in the rat, neither chronic ethanol consumption nor elevated acetaldehyde induces changes in transferrin microheterogeneity.

Comparison of different methods for detecting carbohydrate-deficient transferrin.

Different methods for detecting carbohydrate-deficient transferrin (CDT) were compared. In addition, their efficiency for detecting alcohol abuse among men not having clinical evidence of liver disease was studied in controls (n = 26), weekend (n = 16) and daily (n = 12) heavy drinkers, and alcoholics (n = 28). Comparisons were made between anion-exchange separation of iron-saturated transferrin (Tf) by microcolumns (CDTect) and by the Fast Protein Liquid Chromatography (FPLC% and FPLC-MG), followed by double-antibody radioimmunoassay of collected fractions. Tf fractions with pl > or = 5.7 were also measured by two different isoelectric focusing (IEF) methods, followed by immunofixation (SA-IEF-CDT and IEF-CDT-TOT), the latter method being used also for detection of asialotransferrin (IEF-CDT-AS). The cut-off was 20 units/liter for CDTect, 4.4% of total Tf for SA-IEF-CDT, and the mean +2 sd of the control group for FPLC-MG (as mg/liter of Tf), FPLC-%, IEF-CDT-TOT, and IEF-CDT-AS (all as percentage of Tf). The overall accuracies (combining sensitivity and specificity) for detecting heavy drinkers of CDTect, FPLO (mg/liter), FPLC (%), SA-IEF-CDT, IEF-CDT-TOT, and IEF-CDT-AS were 63%, 59%, 61%, 74%, 57%, and 63%, respectively; for detecting alcoholics, 87%, 83%, 81%, 89%, 37%, and 76%, respectively. In conclusion, the methods were in rather good agreement with each other. Diagnostic characteristics among heavy drinkers and correlations between methods differed slightly, probably depending on the ability of different methods to separate and detect asialo-, monosialo-, and disialotransferrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate-deficient transferrin as an alcohol marker among female heavy drinkers: a population-based study.

Carbohydrate-deficient transferrin (CDT) has previously been reported to be an excellent marker of male alcoholics. Less is known of its efficiency among women and especially of early-phase alcohol abuse in nonselected populations. The present population-based study examined the diagnostic value of CDT among consecutive women, including 13 teetotallers, 135 social drinkers (mean alcohol consumption 45 +/- 34 g/week), and 57 nonalcoholic heavy drinkers (197 +/- 97 g/week). Sixty-two women with a well-documented history of chronic alcoholism (942 +/- 191 g/week) were also studied, as well as 36 pregnant women used as a reference group. Two weeks of abstinence among 11 alcoholics was followed. The CDT (containing part of isotransferrin with pI = 5.7, 5.8, and 5.9) was separated by anion exchange chromatography and assayed by radioimmunoassay. In the whole material, CDT correlated significantly with alcohol consumption (r = 0.43, p < 0.001) but not with conventional markers (gamma-glutamyltransferase, AST, ALT, and mean corpuscular volume). The CDT values of alcoholics (34 +/- 20 units/liter) were significantly (p < 0.001) higher than those of teetotallers (19 +/- 6 units/liter), social drinkers (20 +/- 6 units/liter), or pregnant women (16 +/- 3 units/liter). Heavy drinkers also had higher values (25 +/- 13 units/liter), but the difference did not reach statistic significance. The specificity of CDT was on the level of conventional markers when the cut-off value was increased from 26 to 29 units/liter. At a specificity of 95%, CDT found 19% of the heavy drinkers and 52% of the alcoholics; the best traditional marker, AST, with a specificity of 97%, found 7% and 56%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood tests for detection of alcoholic cause of acute pancreatitis.

We investigated the ability of various blood markers to detect an alcoholic cause of acute pancreatitis. Serum carbohydrate-deficient transferrin (CDT) was significantly correlated with reported 2 month and 7 day ethanol consumptions and was significantly higher in 42 patients with alcoholic acute pancreatitis and in 24 patients with possibly alcoholic acute pancreatitis than in 20 patients with non-alcoholic disease. At a cutoff over 17 U/L, the specificity of CDT was 100% and the sensitivity was 75% to detect an alcoholic cause of acute pancreatitis. The lipase/amylase ratio index, erythrocyte mean corpuscular volume, and gamma glutamyl transferase could not distinguish alcoholic from non-alcoholic acute pancreatitis.

Hidden alcohol abuse among women.

Seven years ago the authors examined 64 consecutive female out-patients with macrocytosis (erythrocyte mean cell volume > or = 100 fl). The cause remained undetermined in 23 (35.9%). Their patient histories were evaluated in 1992, and the new highly specific alcohol marker, serum carbohydrate-deficient transferrin, from the frozen sera taken during the initial study was examined. It was elevated in 6 of the 23 women. Furthermore, four of these six had visited the health centre after 1985 and they all had clinical records indicative of alcohol problems. Because early intervention has proved to be effective, information about the risks of alcohol abuse (intervention) should be given to women with suspect alcohol-induced symptoms or signs.

Amount of alcohol is an important determinant of the severity of acute alcoholic pancreatitis.

BACKGROUND: Does the amount of recently consumed alcohol correlate with the severity of acute alcoholic pancreatitis? METHODS: One hundred one consecutive episodes of acute pancreatitis (AP) were prospectively studied. Seventy-three were alcoholic AP episodes; 40 patients had their first alcoholic AP episode. A standard personal interview was used to determine the alcohol consumption during 2 months and during 1 week before AP. The severity of AP was evaluated according to the Ranson criteria, the serum C-reactive protein (CRP) concentration measured 24 to 48 hours after admission, the length of the hospital stay, the development of complications, and the mortality rate. RESULTS: In the 40 patients having their first alcoholic AP episode, the reported 2-month alcohol consumption correlated significantly with the number of positive Ranson criteria (correlation coefficient r = 0.44, p < 0.01), serum CRP concentration (r = 0.51, p < 0.001), and the length of the hospital stay (r = 0.45, p < 0.01). Complications occurred in eight of 14 patients with 2-month alcohol consumption of more than 5000 gm as compared with one of 14 patients with consumption of less than 2000 gm (p < 0.05). In the same 40 patients the 1-week alcohol consumption correlated with the number of positive Ranson criteria (r = 0.40, p < 0.05) and serum CRP concentration (r = 0.37, p < 0.05). Of the 12 patients who had consumed more than 1000 gm alcohol during the last week before admission, two died and complications developed in six (50%), as compared with none (p < 0.05) and six (21%), respectively, of those who had consumed less than 1000 gm. No significant correlations were observed between the reported alcohol consumption and any of the severity parameters in the 33 patients with recurrent episodes of alcoholic AP. CONCLUSIONS: The amount of alcohol consumed may be an important determinant of the severity of the first alcoholic AP episode but not of recurrent alcoholic AP.

Semi-automatic method for determination of different isoforms of carbohydrate-deficient transferrin.

Carbohydrate deficient transferrin (CDT) has been reported to be one of the best biochemical markers of alcohol abuse. However, a need still exists for a simple and practical method for widespread laboratory use. A semi-automatic (SA) isoelectric focusing (IEF) assay for CDT (SA-IEF-CDT) by a Phast System is introduced here. Different isoforms of transferrin were separated by IEF on polyacrylamide gels (pI 4.0-6.5) and located by immunofixation with an anti-transferrin serum. The precipitation bands were stained with Coomassie Brilliant Blue and quantitated densitometrically. The present method gave a picture of the relative amounts of 10 different transferrin isoforms. The percentage of CDT with pI > or = 5.7 (representing di-, mono- and asialotransferrin) was calculated. For comparisons transferrin bands with pI > or = 5.6 (tri-, di-, mono-, and asialotransferrin), pI > or = 5.8 (mono- and asialotransferrin) and pI > or = 5.9 (asialotransferrin) as well as GGT, ASAT and ALAT were calculated. The method showed good linearity and it identified different isoforms in concentrations of < 10 mg/l of transferrin. The correlation of the present method with a commercially available method employing anion exchange followed by double antibody RIA (AE-RIA-CDT) was good (n = 38, r = 0.924). In 19/20 (95%) of healthy controls, the CDT value was below 4.4% (mean + 2 S.D.) of total transferrin, while higher values were observed in all 20 (100%) alcoholics. In conclusion, the developed semi-automatic method is a practical and reliable alternative for determination of different transferrin isoforms.

CDT by anion-exchange chromatography followed by RIA as a marker of heavy drinking among men.

Carbohydrate-deficient transferrin, CDT, had previously been reported to be an excellent marker for alcoholism. The present population-based study examined the diagnostic value of CDT among consecutive middle-aged males including 122 social drinkers (mean alcohol consumption 88 +/- 79 g per week) and 77 non-alcoholic heavy drinkers (301 +/- 195 g/wk). Ninety-six men with a well-documented history of chronic alcoholism (> or = 1000 g/wk) were used as a reference group. The CDT (containing mainly isotransferrin with pI = 5.8 and 5.9) was separated by anion exchange chromatography and assayed by RIA. The CDT values of social drinkers (mean +/- SD = 14 +/- 5 U/I) were significantly lower than those of heavy drinkers (19 +/- 13 U/I, p < 0.01) and alcoholics (34 +/- 18 U/I, p < 0.001). In the whole material CDT correlated positively with alcohol consumption (r = 0.53, p < 0.001). At a specificity of 91.8%, CDT found 28.6% of the heavy drinkers and 79.2% of the alcoholics; the best traditional marker, GGT, with a specificity of 86.9%, found 35.1% and 64.6%, respectively. In conclusion, CDT is a specific marker, which is superior to traditional markers for identifying alcoholics. Unfortunately, it does not seem to provide additional power for identifying the important group, non-alcoholic heavy drinkers.