In vivo study on lead and alcohol interaction and the inhibition of erythrocyte delta-aminolevulinic acid dehydratase in man.

S. In vivo study on lead and alcohol interaction and the inhibition of erythrocyte delta-aminolevulinic acid dehydratase in man. Scand J Work Environ Health 10 (1984) 239-244. The effect on alcohol (EtOH) consumption on the inhibition of erythrocyte 6-amino- levulinic acid dehydratase (ALAD) was investigated in 13 male lead workers and 7 "normal" male subjects. Lead and zinc protoporphyrin in blood and lead, 6-aminolevulinic acid, porphobilinogen and coproporphyrin in 24-h urine specimens were also determined. During 1 h the subjects drank 122.8 (SD 18.65) ml of an almost lead-free brandy, ie, a dose of 11.07 mmol/kg of body weight. This dose resulted in a trend toward a parallel decrease in ALAD activity and an increase in lead in blood (PbB), both of which approached the prealcohol value 24 h after the initial alcohol ingestion. A trend toward increased lead excretion in urine (PbU) was observed on the day of alcohol ingestion, as compared to the preceding and succeeding 24-h urine specimens. However, the observed increase in PbB and PbU cannot be attributed to the small amount of lead ingested through the brandy, ie, 7.09 (SD 1.06) nmol. The characteristic dose-effect relationship between PbB and ALAD (examined prior to and 1, 3, 5, and 24 h after the initial alcohol ingestion) reached the highest correlation coefficient 3 h after the initial alcohol ingestion (p < 0.001). The data obtained appear to support the hypothesis of a possible role for the body lead pool and the lead-mediated influence of alcohol consumption on ALAD activity in man. of lead exposure, biologically active lead deposit, ethanol.

The results of our previous study (17) have indicated that ethanol (EtOH) per se does not inhibit the activity of erythrocyte 6-aminolevulinic acid dehydratase (ALAD) in human blood in vitro. However, it has been confirmed that ALAD activity is readily inhibited by lead. These findings were the basis for the present in vivo study, which was carried out with the same objective: to corroborate the assumption of the possible role of the body lead pool in the inhibition of ALAD activity following alcohol ingestion.

Subjects and methods
The study was performed with two groups of male volunteers: 13 subjects occupationally exposed to lead (7 from a ceramics factory and 6 from a lead products factory) and 7 "normal" subjects nonoccupationally exposed to lead. The subjects were given 122.8 (SD 18.65) ml of Trenk brandy (40 volume/ volume% EtOH) to drink during a period of 1 h, ie, an ethanol dose of 11.07 mmol/kg of body weight, which is equivalent to a peak ethanol concentration of approximately 16.28 mmol/l of blood, known to produce a slightly drunken state (16). Our previous data on blood ethanol determination (after the ingestion of the same dose of Trenk brandy under identical conditions as in the present study) have indicated that a maximal blood ethanol concentration close to 16.28 mmol/l of blood is reached in the period 1-2 h after the initial ingestion of the brandy, while it is considerably lowered (to approximately 7.60 mmol/l blood) in the period 3 h after the commencement of the experiment (16).
The concentrations of lead and zinc in the brandy were measured with atomic absorption spectrophotometry (AAS) (electrothermal AAS technique for lead and flame AAS technique for zinc). The Trenk brandy contained 57.9 nmol of lead and 4.36 pmol of zinc per liter of brandy. Therefore the amount of lead ingested through the brandy corresponded to 7.09 (SD 1.06) nmol of lead, which is a negligible amount if one considers the low lead absorption by the ingestion (about 10 Yo) and dilution of lead in the whole body blood volume (4-5 1). The concentration of zinc in the brandy was lower than in tap water.  In both groups of subjects, the ALAD activity (2) and lead in blood (PbB) (7) were determined immediately before the ingestion of the brandy (hour 0) and at 1-, 3-, 5-, and 24-h intervals after the first measurement. The concentration of zinc protoporphyrin (ZnPP) in blood (3) was measured before the brandy ingestion and 24 h later. Urine (a 24-h specimen) was collected on three consecutive days, ie, a day before brandy consumption (day 0), on the day of consumption (day I), and the day after consumption (day 2).
An extra precaution was taken to ensure the correct collection of 24-h urine specimens. (The lead workers were hospitalized in our department and were under continuous control during the period of investigation, while the "normal" subjects were our colleagues from the Institute and were fully aware of the problem.) In each urine specimen the concentrations of lead (PbU) (24), 6-aminolevulinic acid (ALAU) (6), porphobilinogen (PBGU) (12), and coproporphyrin (CPU) (23) were measured. The dithizone method used for determining PbU (24) was modified for the urine analysis, ie, by an increase in the sample volume (50 ml of urine), a gradual addition of nitric acid (5-ml aliquots), and the calibration performed for the urine. The precision of the applied methods, expressed as a relative standard deviation (CV), was I 5 % for PbB, 2 % for ALAD, 5 5 % for ZnPP, 5 4 % for PbU, ALAU and PBGU, and 5 5 % for CPU. All the analyses were performed in duplicate, and the mean values were used for the calculations.
The results of the biological indicators following alcohol ingestion were compared to prealcohol values, and the significance of the difference was tested by Student's t-test (ie, t = &,,,/sE,,,J. Due to the possible influence of diuresis, the data on the urinary indicators (PbU, ALAU, PBGU, CPU) have been expressed both in terms of daily excretion (quantity excreted per day) and the concentration (quantity per liter of urine).

Results
The data on the simultaneous determination of the biological indicators of lead absorption (PbB, ALAD, ZnPP, PbU, ALAU, PBGU, CPU), measured before alcohol consumption and at different periods following alcohol ingestion (expressed as the difference between post-and prealcohol values), are presented in tables 1-3.
The relative changes in ALAD and PbB, expressed as the ratio between the post-and prealcohol values, are shown in figure 1. These data indicate that the ingestion of the alcoholic beverage resulted in a depression of ALAD activity and a parallel increase in PbB. The change in both parameters appeared to reach a maximum in the period 3 h after the initial alcohol ingestion. Both PbB and ALAD approached the prealcohol value 24 h after the initial alcohol ingestion.
Due to the small number of subjects and considerable inter-and intraindividual variations in diuresis after alcohol consumption, eg, decreased diuresis in lead workers and increased diuresis in "normal" subjects (table 2), the results concerning the urinary indicators are expressed as the quantity per liter of urine (table 3) for a better comparison of the data of both groups. These data indicate an increase in PbU on the day of alcohol ingestion (day I), as compared to preceding and succeeding 24-h urine specimens. However, although an identical dose of alcoholic beverage was given (providing a comparable increase in both EtOH and Pb), the increase in PbU appeared to be considerably more pronounced for the lead workers as compared to the "normal" subjects.
The semilogarithmic dose-effect relationship between PbB and ALAD was highly significant (p < 0.001) for all the examined intervals, ie, log ALAD/lin PbB (N = 20): r, = -0.878, r, = -0.817, r, = -0.887, r, = -0.751, r,, = -0.826. However, for certain periods, the relationship was less significant within the group of lead workers (N = 13): r, = -0.801, (p < 0.001), r, = -0.681 (p < 0.02), r, = -0.855 (p < 0.001), r, = -0.549 (p > 0.05), r,, = -0.655 (p < 0.02), and not significant at all (p > 0.10) within the group of "normal" subjects (N = 7). The highest correlation coefficient (r) was recorded 3 h after the initial alcohol ingestion. The relationship between PbB and ALAD for lead workers and for the pooled data of both groups is presented for the period immediately before alcohol ingestion (figure 2), 3 h later (figure 3), and 24 h after the first measurement ( figure 4). The differences in dose-effect relationship between PbB and ALAD with respect to alcohol consumption appear to support the hypothesis that the changes in both parameters reflect the alcohol-induced redistribution between the "stable" and the "mobile" lead frac-  sequent partial excretion of the mobilized lead.

Discussion
The present data indicate the possibility of a leadmediated influence of alcohol consumption on the inhibition of erythrocyte ALAD activity in man. This phenomenon is seen in the trend towards increasing PbB, occurring in parallel with a decrease in ALAD, in a lead-exposed and "normal" group following alcohol ingestion ( figure 1). Moreover, the doseeffect relationship between PbB and ALAD reached the highest correlation coefficient at a time when the highest blood ethanol concentrations were no longer expected (16) but the lowest ALAD activity and the highest PbB values were observed. In addition an increase in PbU was found in both groups on the day of alcohol ingestion, as compared to preceding and succeeding 24-h urine specimens (tables 2 & 3). Since the observed increase in PbB and PbU cannot be attributed to the relatively negligible intake of lead through the brandy, the possibility of an alcoholinduced interchange between the "stable" and "mobile" lead fractions of the total body lead pool (eg, through alcohol-induced alterations in the tissue redox potential) should be taken into consideration. With regard to the application of ALAD as a biological indicator of lead exposure (absorption), the influence of alcohol consumption on erythrocyte ALAD in man was usually interpreted as an interference which decreases the specificity of ALAD for lead. A significant negative correlation between the ALAD and ethanol levels in the blood was found for patients intoxicated with alcohol (15). However, no significant correlation between ALAD and ethanol has been found by other authors (9, 11). A decrease of erythrocyte ALAD has often been associated with an increase of PbB (1 1, 14, 22), and highly significant correlations between ALAD and PbB have also been reported (1 1, 22). Clinical experience has indicated that alcohol consumption may be a predisposing factor for lead poisoning (4,5). In addition to decreased levels of ALAD, the findings reported with respect to alcohol consumption (13) appear to resemble closely the characteristic effects of lead.
The decreased ALAD levels found to be associated with alcohol consumption can be due to increased lead intake (through lead-contaminated alcoholic beverages) and/or increased lead mobilization (ie, activation of the "stable," biologically inert lead fraction of the total body lead pool) with a transient increase in the bone marrow lead, and in PbB, and subsequent partial excretion. Since the former possibility is indisputable, with regard to the latter possibility, it is interesting to note that even slightly higher than average "normal" ALAD values were found in chronic alcoholics during the inactive phase (10, 11).
T h e latter possibility might partly explain why the observed increase of P b B with respect t o alcohol consumption in a n urban population could not be ascribed t o any particular form of alcoholic beverage (ie, to the differences in lead content), but rather the total alcohol intake seems t o be relevant (8,19). It might also explain why a significant increase in P b B (r = 0.296, p < 0.02) a n d a decrease i n ALAD (r = -0.255, p < 0.05) was found with respect to the age of 66 patients with nonalcoholic liver disease, a n d an opposite trend with respect to age (although not significant) was found for both P b B (r = -0.029) a n d ALAD (r = 0.118) for 211 patients with alcoholic liver d i~e a s e .~ I n both groups highly significant @ < 0.001) correlations between ALAD and P b B were found (r = -0.408 for nonalcoholics a n d r = -0.516 for alcoholics). However, even the lack of a significant correlation between ALAD a n d P b B (14) should not autolhatically exclude the possible role of lead in the depression of ALAD activity. It has been shown that P b B mainly reflects t h e current (recent) lead exposure level, while ALAD better reflects the integrated (long-term) lead exposure and the biologically active lead deposit (1,20,21). Therefore the possible influence of alcohol consumption via a biologically active lead deposit, which is less reflected by P b B than by ALAD, can even be regarded as a possible explanation for such findings.
In conclusion, the present results indicate that data on the specificity of ALAD for lead, ie, interference of other factors o n ALAD activity in vivo, should be interpreted with caution. However, the reported influence of, eg, alcohol consumption (10, 14, 15) o r smoking (18) o n the depression of ALAD activity in m a n might also be explained via a biologically active lead deposit, less reflected by PbB t h a n by ALAD.