Urinary ALA Research Articles

Fluorometric HPLC determination of delta-aminolevulinic acid (ALA) in the plasma and urine of lead workers: biological indicators of lead exposure.

A fluorometric high-performance liquid chromatographic (HPLC) method was developed for the highly sensitive measurement of delta-aminolevulinic acid (ALA) in biological materials. By using this method, we determined ALA in the plasma and urine of 418 workers occupationally exposed to lead and in the plasma and urine of 227 controls. The concentrations of ALA in the plasma and urine of lead workers were significantly elevated as compared with those of the controls. The concentration of ALA in plasma and urine was highly correlated with that of lead in blood in lead workers. It was found that the correlation (r = 0.742) between log of plasma ALA concentrations and blood lead concentrations in lead workers was similar to that (r = 0.711) between log of urine ALA concentrations and blood lead concentrations. These results demonstrated that the measurement of ALA in plasma or in urine using a fluorometric HPLC method was useful for the biological monitoring of lead workers.

Indices of lead-exposure in blood and urine of lead-exposed workers and concentrations of major and trace elements and activities of SOD, GSH-Px and catalase in their blood.

Seventy male factory workers were studied. The lead concentrations in their blood (Pb-B) were 16.55 +/- 11.53 micrograms/100 ml (range 1.5 to 50.2 micrograms/100 ml). The subjects were divided into three groups according to Pb-B (in microgram/100 ml): group A, Pb-B < or = 10 (n = 22); group B, 10 < Pb-B < or = 20 (n = 30); group C, Pb-B > 20 (n = 18). The mean +/- S.D. in each group was 5.57 +/- 2.53, 15.02 +/- 2.75, and 32.52 +/- 9.49 micrograms/100 ml, respectively. Pb in plasma was 0.011 +/- 0.010, 0.017 +/- 0.033, and 0.021 +/- 0.021 microgram/liter, and Pb in the RBC was 0.281 +/- 0.246, 0.701 +/- 0.325, and 1.626 +/- 0.861 micrograms/g Hb, respectively. In addition to Pb concentration, the concentrations of 34 elements in the plasma or in the RBC were determined. Se concentrations in RBC in each group were 0.618 +/- 0.139, 0.670 +/- 0.207, and 0.728 +/- 0.200 microgram/g Hb, and the mean values were significantly different between groups A and C (p < 0.05). For Se concentration in plasma, the mean +/- S.D. in each group was 0.132 +/- 0.035, 0.130 +/- 0.031, and 0.126 +/- 0.021 microgram/ml, respectively, and there was no significant difference between groups. On the other hand, when the activities of total SOD, Mn-SOD, Cu, Zn-SOD, and catalase in the plasma and the activities of GSH-Px both in the plasma and in the RBC were assayed, some differences were found. The activities in GSH-Px in RBC were 17.19 +/- 5.03, 17.59 +/- 3.95, and 15.25 +/- 3.18 mumol/g Hb/min, and those in plasma were 0.069 +/- 0.032, 0.081 +/- 0.023, and 0.080 +/- 0.028 mumol/ml/min. In group C, GSH-Px activity was lower in the RBC and higher in the plasma than those in group A, and it was observed that the Se concentration was higher in RBC, and that there was no remarkable change in the plasma. Catalase activity in group C was 3.58 +/- 0.81 mgH2O2/ml/30 min, which was significantly higher than that in group A (2.81 +/- 0.90 mgH2O2/ml/30 min). Further investigation is necessary in order to explain the above results. The regular indices used for evaluating lead exposure, showed significant correlations with Pb-B: r = -0.786 vs delta-Aminolevulinic acid (ALA) dehydratase activity in blood, r = 0.927 vs. inhibition rate, and r = 0.339 vs. ALA in urine.

Improvement of urinary delta-aminolevulinic acid determination by HPLC and fluorescence detection using condensing reaction with acetylacetone and formaldehyde.

We improved the method for determining urinary delta-aminolevulinic acid (ALA) by HPLC-fluorometer after pre-column derivatization with acetylacetone and formaldehyde, and a stable ALA derivative was obtained without any effect from various urinary components as demonstrated by the complete recovery of ALA (100.9 +/- 5.5%, n = 85) from the urine samples. The modified procedure was as follows: Twenty microliters of urine sample, 5 ml of acetylacetone solution (acetylacetone/ethanol/distilled water containing 4 milligrams of NaCl; 15/10/75), and 0.45 ml of 9.3% formaldehyde solution were mixed and boiled for 15 min. The fluorescent derivative of ALA was separated and analyzed by HPLC with the fluorometer at Ex 246 nm and Em 458 nm. Using a gradient program, the retention time of the ALA derivative was 7.3 min and the analysis could be repeated at 13 min intervals. Concentrations of ALA in urine samples measured by this method were significantly correlated with those measured by the Mauzerall-Granick (M-G) method (n = 85, r = 0.993, p < 0.001). The values obtained by our method were, however, lower than those obtained by the M-G method. Urinary ALA concentrations of 40 non-lead workers ranged from 0.1 to 2.3 mg/g creatinine with the mean +/- SD of 1.1 +/- 0.4 mg/g creatinine as measured by the present method.

Postlabeling Analysis of DNA Adducts in White Blood Cells from Lead Smelter Workers.

We analyzed DNA adducts levels in white blood cells from 14 lead smelter workers for the first time as an indicator of the effect of lead exposure and compared with some lead exposure indices to evaluate whether lead exposure induces DNA adducts significantly. DNA adducts were measured by the nuclease P1 enhanced 32P-postlabeling method. As the lead exposure indices, we chose blood lead (Pb-B), urinary delta-aminolevulinic acid (ALA) and erythrocyte protoporphyrin (FEP). The levels of DNA adducts had a weak positive correlation with urinary ALA concentration (r = 0.62), but not significant correlation with Pb-B and FEP. This result suggests that lead exposure might have the effect to remain DNA adducts. The inhibition of DNA repair system may be one reason. This preliminary study need be followed by extended surveys on lead exposure.

Usefulness of delta-aminolevulinic acid in blood as an indicator of lead exposure

The concentration of delta-aminolevulinic acid in blood (ALA-B) was determined using high performance liquid chromatography (HPLC). To improve the chromatographic separation and the recovery rate of ALA determination in blood, acetate buffer was used in the reaction mixture of fluorescence derivatization. The detection limit of ALA-B was ca. 2 micrograms/l at signal to noise ratio of 5, and the analytical recovery was 102.0 +/- 4.10% (mean +/- SD), when 50 micrograms/l of ALA was added to 7 blood samples (ALA-B levels: 6.5-103.0 micrograms/l). ALA-B levels in control subjects (n = 19) were 5.3 +/- 1.4 micrograms/l (mean +/- SD) and those in 52 lead workers (blood lead levels (Pb-B): 2.4-86.2 micrograms/dl) were 15.4 +/- 12.2 micrograms/l (range: 3.1-137.3 micrograms/l). Standard curve of ALA was linear over a wide range, at least up to 400 micrograms/l. In the workers, the correlation coefficients of ALA-B vs. Pb-B and ALA-B vs. delta-aminolevulinic acid dehydratase activity (ALA-D) were higher than those of urinary concentration of ALA vs. Pb-B and that vs. ALA-D, especially in the moderate Pb-B level (less than 40 micrograms/dl, n = 35). Unless the urinary concentrations of ALA were not corrected, significant correlation could not be found between Pb-B and urinary ALA in the workers moderately exposed to lead, and the correlation coefficient between urinary ALA and ALA-D was -0.354, while the correlation coefficients of ALA-B vs. Pb-B and ALA-B vs. ALA-D were 0.739 and -0.746, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Interrelation between urinary delta-aminolevulinic acid (ALA), serum ALA, and blood lead in workers exposed to lead.

Using a fluorometric HPLC method, we determined delta-aminolevulinic acid (ALA) in sera and urine samples from 16 lead workers with blood lead levels ranging from 19 to 107 microG/100 ml. The concentration of ALA in serum correlated highly with the urinary ALA concentration (gamma = 0.957 for ALA mg/1; gamma = 0.967 for ALA mg/g creatinine). The ALA concentrations in the serum of lead workers ranged from 11 to 151 micrograms/1 with a mean of 51 micrograms/1. In addition, the concentrations of urinary ALA (mg/g creatinine) and serum ALA micrograms/1) had a strong correlation with blood lead concentrations (gamma = 0.838 and 0.892, respectively). These data indicate that the measurement of serum ALA, as well as urinary ALA, is very useful for the biological monitoring of occupational lead exposure.

Liver δ-aminolevulinate dehydratase activity in amitriptyline- or chlorpromazine-treated rats

Amitriptyline (AMT) and chlorpromazine (CPZ) (0.5 mg per animal, i.p.) were injected into rats separately for 30 days and their effects on heme metabolism in liver were examined. Significant decreases in the δ-aminolevulinate dehydratase activity were observed following the administration of both drugs (mean value of AMT-group: 6.58 U/g tissue; and CPZ-group: 7.04 U/g tissue) in comparison to that of controls (11.71 U/g tissue); however total liver heme content was not altered. When 24-h urinary excretions of δ-aminolevulinate (ALA) and porphobilinogen (PBG) were measured on the last day of the experiment, a slight (AMT-group: 38.40 μg/day) to distinct (CPZ-group: 59.11 μg/day) increase of urinary ALA was observed, while PBG excretion tended to decline only moderately under CPZ (3.52 μg/day), but significantly in presence of AMT (2.16 μg/day). Mean values obtained from control group were 32.12 μg/day for ALA and 4.25 μg/day for PBG.

Direct injection method for quantitation of delta-aminolevulinic acid in urine by high-performance liquid chromatography.

A highly sensitive and simple method for determining delta-aminolevulinic acid (ALA) in urine was established, using direct injection of urine into a high-performance liquid chromatographic column, with fluorometric detection after post-column derivatization with o-phthalaldehyde (OPA). The recovery of ALA was about 100% and ALA was completely separated on an ion exchange column (retention time, 38 min). The detection limit for ALA was 10 pmol (S/N = 2). The mean levels of urinary ALA of 10 healthy volunteers, 4 patients with acute intermittent porphyria, and 2 workers occupationally exposed to lead were 0.76, 5.25, and 23.54 mg/l, respectively. Because of its simplicity, the method is considered to be suitable for routine analysis of urinary ALA in the clinical laboratory.

Clinical relevance of urinary delta‐aminolevulinic acid/logarithm of creatinine ratio in screening for occupational lead exposure

Using receiver operating characteristics (ROC) analysis, the clinical relevance of urinary delta-aminolevulinic acid concentration adjusted to the logarithm of creatinine concentration (ALA/log.Cn) proved to be better than that of uncorrected ALA or ALA/Cn ratio for the detection of blood lead levels (Pb-B) exceeding 2.5, 3.0, or 3.5 mumol/l in 483 men occupationally exposed to lead. Ala/log.Cn yielded validities of 1.63, 1.75, and 1.79 at Pb-B levels 2.5, 3.0, and 3.5 mumol/l, respectively. The same values for ALA/Cn were 1.59, 1.70, 1.78 and for ALA 1.52, 1.65, 1.72, respectively. The simple adjustment to log. Cn makes urinary ALA determination more efficient in biological monitoring of the effect of occupational lead exposure.

151 HEMIARGINATE THERAPY FOR PORPHYRIA CRISES OF TYROSINEMIA

Porphyria-like symptoms are common in hereditary tyrosinemia (HT). This is due to inhibition of δ-aminolevulinate (ALA) dehydratase (D) by succinylacetone (SA). We present our experience with hemiarginate (HA) for porphyria crises in two pts with HT. Both showed low activity of erythrocyte ALA-D and sustained high urinary excretion of ALA and SA. The symptoms started at 18 mo (Pt 1) and 3 yrs (Pt 2) with irritability, drowsiness, vomiting, hypertension, and peripheral neuropathy. ALA excretion increased up to 15 times the upper normal limit. When intravenous HA was given (for 4 d), the pts became alert within the first day, painless and normotensive within 3 d, and the neuropathy vanished within 2 wks. Urinary ALA excretion decreased within 24 h. In all, 10 relapses occurred and were treated with HA. In Pt 1 prophylactic HA twice a wk for up to 9 mo prevented recurrences. No side effects of HA were noted. Conclusion: HA is a safe and effective therapy for porphyria crises of HT.

Comparison of hepatic .DELTA.-aminolevulinic acid(ALA) synthase and ALA dehydratase activities and urinary ALA excretion in different strains of male mice.

Comparison of hepatic .DELTA.-aminolevulinic acid(ALA) synthase and ALA dehydratase activities and urinary ALA excretion in different strains of male mice.

Tolerance to lead-induced porphyrin metabolic disorders following lead pretreatment in mice

The protective effect of pretreatment with lead on lead-induced toxicity was investigated in mice, using some biological parameters such as urinary excretion of δ-aminolevulinic acid (ALA) and coproporphyrin, accumulation of erythrocyte protoporphyrin and inhibition of erythrocyte (5-aminolevulinic acid dehydratase; these are useful indicators for evaluating the effects on health of lead. It was demonstrated that pretreatment with a single intraperitoneal dose of 2 mg Pb kg , 7 days prior to the challenge dose, prevents in part the increasing excretion of urinary ALA induced by a challenge exposure to lead (200 ppm) in the drinking water for 7 days.

Studies on erythrocyte pyrimidine 5?-nucleotidase (P5N) test and its evaluation in workers occupationally exposed to lead

An erythrocyte pyrimidine 5'-nucleotidase (P5N) test was performed for 171 workers occupationally exposed to lead. Erythrocyte P5N activity was markedly inhibited by exposure to lead. Among several biological indicators (erythrocyte P5N, delta-aminolevulinic acid dehydratase (ALAD), protoporphyrin (PROTO), urinary delta-aminolevulinic acid (ALA), coproporphyrin (COPRO)), the P5N activity had the highest correlation with the concentration of lead in blood (r = -0.77). A significant inhibition of erythrocyte P5N was found in groups of lead workers with blood-lead levels of more than 10 to 19 micrograms/dl. This P5N inhibition started before any changes occurred in urinary ALA and COPRO. A 45 to 50% inhibition of P5N corresponded to the blood-lead value (50 micrograms/dl) of the BEI recommended by ACGIH. In some lead workers, erythrocyte nucleotides (mainly CTP and UTP) were determined. The data indicated that a marked accumulation of these nucleotides had occurred, and their levels correlated negatively with P5N activity and positively with blood lead.

Relationship between inhibition of erythrocyte pyrimidine 5'-nucleotidase activity and biological response for porphyrin metabolism in workers occupationally exposed to lead.

A rapid determination of erythrocyte pyrimidine 5'-nucleotidase (P5N) activity in lead workers was carried out using a high-performance liquid chromatograph (HPLC). The P5N activity had a good negative correlation with the concentration of lead in blood (PbB) ranging from 16 to 96 micrograms/dl (r = -0.82, n = 77). Further, the P5N was compared with other biological parameters: erythrocyte delta-aminolevulinic acid dehydratase (ALAD) activity, erythrocyte protoporphyrin (PROTO), urinary delta-aminolevulinic acid (ALA) and urinary coproporphyrin (COPRO). The correlation coefficients between P5N and ALAD, log PROTO, log ALA, and log COPRO were 0.59, -0.72, -0.65, and -0.61, respectively. On the other hand, the normal value of P5N obtained from 72 healthy subjects was 11.9 +/- 2.1 units; mumol uridine/h/g Hb (mean +/- SD), indicating that the lower limit of 95% confidence interval for normal P5N was about 8 units. When P5N was cut off at less than or equal to 8 units in 77 lead workers, the validity (sensitivity + specificity) for PbB greater than or equal to 40 micrograms/dl, PbB greater than or equal to 60 micrograms/dl, erythrocyte PROTO greater than or equal to 150 micrograms/dl RBC, urinary ALA greater than or equal to 6 mg/l, and urinary COPRO greater than or equal to 150 micrograms/l was 1.66, 1.76, 1.57, 1.68, and 1.60, respectively. From these results, it was confirmed that the erythrocyte P5N test is suitable for the biological monitoring of exposure to lead in a wide range, and its activity is useful in predicting the disturbance of porphyrin metabolism induced by lead.

Species difference of urinary excretion of δ-aminolevulinic acid and coproporphyrin in mice and rats exposed to lead

The urinary excretion of δ-aminolevulinic acid (ALA) was first investigated in mice exposed to lead in the drinking water (500 ppm) for 30 days, and the results were compared with those obtained from rats exposed to lead under the same exposure condition. The result indicated that the level of urinary ALA excretion in lead-exposed mice was much higher than that in lead-exposed rats. This finding suggests a species difference between mice and rats with regard to sensitivity to lead. On the other hand, urinary coproporphyrin (CP) was also determined in rats and mice exposed to lead. The result obtained indicated that the increasing level of urinary CP excretion was higher in rats than in mice, contrary to expectation. The reason for the discrepancy between urinary excretion of ALA and that of CP in rats and mice exposed to lead is, however, unknown.

Urinary excretion of 5-hydroxyindoleacetic acid, δ-aminolevulinic acid and coproporphyrin isomers in rats and men exposed to lead

The urinary excretion of 5-hydroxyindoleacetic acid (5-HIAA), δ-aminolevulinic acid (ALA) and coproporphyrin (CP) isomers (I and III) was investigated in rats and workers exposed to lead. Urinary excretion of 5-HIAA, which is the major metabolite of serotonin, was not influenced by the lead exposure. However, a significant increase in urinary ALA and CP was found in rats and workers exposed to lead. In particular, the urinary excretion of CP-III was markedly increased compared to that of CP-I in rats and workers exposed to lead.

Optimized liquid-chromatographic method for fluorometric determination of urinary delta-aminolevulinic acid in workers exposed to lead.

We developed a fluorometric method for determining delta-aminolevulinic acid (ALA) in urine of lead workers. A high-performance liquid chromatograph (HPLC) equipped with a fluorescence HPLC monitor is used. The detection limit for aqueous ALA is 20 micrograms/L (15 pmol of ALA in the 100-microL sample). The working linear range of urinary ALA concentration was 0.1 to 100 mg/L. In 25 lead-exposed workers, ALA values by the present method for urine correlated well with those obtained by a conventional colorimetric method (r = 0.996). The advantage of the present method for micro-determination of urinary ALA is its high sensitivity.

Factors affecting determination of delta-aminolevulinate by use of Ehrlich's reagent.

We investigated the formation of delta-aminolevulinic acid (ALA)-pyrrole and subsequent color development used in determining ALA in urine. Of three condensation reagents examined--acetylacetone, ethyl acetoacetate, and methyl acetoacetate--the last was the best. When the ALA-pyrrole is reacted with Ehrlich's reagent in ethyl acetate according to one procedure, the optimum concentration of perchloric acid in the reagent is 0.5 to 1.0 mol/L. Its optimum concentration is 1.0 to 2.3 mol/L when the reaction takes place in water.

Improved method for the determination of urinary delta-aminolevulinic acid in lead workers

An improved method for the determination of urinary delta-aminolevulinic acid (ALA) as an indicator of lead absorption is described. It is a modification of the analytical procedure described by Tomokuni and Ogata in 1972. In the present method, the spot urine from lead-exposed workers which was diluted 3-fold with distilled water beforehand was used as the sample to eliminate as much as possible the urinary matrix interference. In addition, methyl acetoacetate was used as the condensation reagent for ALA-pyrrole, because the color intensity of ALA-pyrrole was higher with the use of methyl acetoacetate than that with ethyl acetoacetate which was used in the original method. The urinary ALA value determined by the present method was similar to that measured using an internal-standard addition method. This method is suitable as a rapid and inexpensive screening procedure for the routine quantitative analysis of urinary ALA.

Lead and zinc concentrations in plasma, erythrocytes, and urine in relation to ALA-D activity after intravenous infusion of Ca-EDTA.

Lead and zinc concentrations in plasma, erythrocytes, and urine, urinary ALA concentration, and ALA-D activity in blood were studied for four hours in two male lead workers during and after a one hour infusion of Ca-EDTA 2Na. Urinary and plasma lead concentrations increased as a result of administering Ca-EDTA 2Na, and the ratios of lead concentrations in plasma to those in urine were greatly increased. The increase of plasma lead concentration was not due to the haemolytic effect of Ca-EDTA 2Na but was mobilised lead, rapidly excreted in the urine. ALA-D activity in blood increased at the end of the experiment with a transient decrease during the infusion of Ca-EDTA 2Na. As zinc concentrations in erythrocytes and plasma did not decrease during the infusion despite an increase in the urinary excretion of zinc, the transient decrease of ALA-D activity was not due to a loss of zinc caused by Ca-EDTA 2Na. From the results of additional experiments in vitro, this transient decrease could be related neither to Ca-EDTA 2Na nor to lead in the blood.