Int Arch Occup Environ Health (1992) 63:419-421
InternationalArchivesof
Oc Upationald
Environmental Itealth © Springer-Verlag 1992
Erythrocyte nucleotides in lead workers Masayoshi Ichiba l , Katsumaro Tomokuni', and Koji Mori 2
'Department 2
of Community Health Science, Saga Medical School, Nabeshima, Saga 849, Japan Nishinihon Occupational Health Science, Yahatahigashi, Kitakyusyu 805, Japan
Received June 25 / Accepted October 10, 1991
Summary We determined erythrocyte nucleotides levels in 22 lead workers with blood lead ranging from 8 to 78 jgldl Their erythrocyte pyrimidine 5'-nucleotidase (P 5N) activity ranged from 19 4 to 3 4z mol uridine/h per g hemoglobin A significant elevation of erythrocyte pyrimidine nucleotides was found, and their contents correlated inversely with erythrocyte P 5 N activity and positively with blood lead concentration In particular, the contents of uridine triphosphate (UTP) and cytidine triphosphate (CTP) exhibited a high correlation with P5N activity (r = -0 56, -0 54) However, its concentration in lead workers was estimated to be much lower than that in hereditary P 5 N deficiency.
In this study we examined the accumulation of specific erythrocyte nucleotides in workers occupationally exposed to lead and compared the data obtained with P 5 N activity and blood lead concentration.
Key words: Lead
Erythrocyte P5 N assay and nucleotides separation The high-performance liquid chromatography (HPLC) system consisted of two LC-9A pumps, SPD-6 AV detector, CTO-6 A column oven, SIL6B autoinjector, SCL-6B system controller, and CR-4 A data processor, all from Shimadzu Co , Kyoto, Japan Erythrocyte P5N activity was determined according to the method of Tomokuni and Ichiba (1986) and expressed as gmol uridine/h per g hemoglobin (Hb). The extraction of nucleotides from erythrocytes was performed as follows: blood (1 ml) was deproteinized by adding 2 ml 0 6 N HC 10 4, and it was centrifuged at 1200 g for 5 min The supernatant (2 ml) obtained was neutralized with 2 ml 0 2 M K2CO 3 The nucleotides were separated and determined by a modification of the method of Angle et al (1982) The extracts (30 gl) were injected onto an anion exchange column (Partisil 5 SAX, 4 6 x 100 mm, 5 Itm particle size; Whatman Inc ) and a gradient elution system (buffer A: 7mMKH 2PO4, pH 3 9; buffer B: 250 mM KH2 PO 4, 500 m M KCI, pH 4 5) was used at a flow rate of 1 5 ml/min An isocratic period of 5 min followed by a 25-min concave gradient from O% to 100 % gave nucleotides separation within 40 min The wavelength was set at 270 nm, and column temperature was set at 50 °C Chromatographic peaks were identified by comparison with reference standards purchased from Sigma Chemical Co.
Nucleotide
Nucleotidase
Introduction Pyrimidine 5 '-nucleotidase (P 5 N, EC 3 1 3 5) specifically dephosphorylates pyrimidine nucleotides Pyrimidine nucleotides resulting from RNA degradation in maturing erythrocytes are normally rendered diffusible by P 5N (Valentine et al 1974) By this mechanism, the maturing erythrocytes retain purine nucleotides as a source of ATP. P 5N activity is a useful indicator of lead exposure (Paglia et al 1975 ; Angle and McIntire 1978 ; Buc and Kaplan 1978 ; Mohammed-Brahim et al 1985 ; Sakai and Ushio 1986 ; Ichiba et al 1987) It was reported that the depression of P 5 N activity results in the accumulation of pyrimidine nucleotides in erythrocytes in clinical studies (Paglia et al 1977 ; Angle et al 1982) and in rabbits treated with lead (Angle et al 1980 ; Swanson et al. 1980) However, there are few reports on workers with occupational exposure to lead (Ichiba and Tomokuni 1990 ; Sakai et al 1990). Offprint request to: M Ichiba
Subjects and methods Subjects Lead-exposed subjects were 22 workers (age 46 ± 11 years) occupationally exposed to lead They were employed in the secondary lead smelter factory Heparinized venous blood samples were collected from them The separation of erythrocytes from whole blood was accomplished by the method of Beutler et al. (1976).
Blood lead analysis The concentration of blood lead was analyzed by flameless atomic absorption spectrophotometry after 10-fold dilution of whole blood with 0 1 NHNO 3 containing 1% triton X-100. Statistical method Data analysis employed Pearson's correlation coefficient.
420 Figure 2 shows the typical chromatograms of erythrocyte nucleotides obtained from a control subject and a lead-exposed worker The peak for uridine triphosphate (UTP) and cytidine triphosphate (CTP), which was undetectable in control subjects, appeared on the lead worker's chromatogram. Table 1 shows the relationship between nucleotides concentration and P5 N activity or blood lead level A significant correlation was found between P 5N activity or blood lead level and the concentration of CTP, UTP or CDP-choline plus CMP The data demonstrated that the occupational exposure to lead elevates the contents of erythrocyte pyrimidine nucleotides, such as CTP, UTP, and CDP-choline plus CMP These results were similar to those reported by Sakai et al (1990) In addition, they reported that the levels of purine nucleotides (ATP, AMP) are affected by lead exposure However, we did not obtain a significant relationship between concentration of purine nucleotides in erythrocyte and lead exposure in the present study. Several investigators have indicated that abnormal accumulation of pyrimidine nucleotides in erythrocytes
Results and discussion Figure 1 shows the correlation between blood lead concentration and erythrocyte P 5 N activity obtained from 22 lead workers The P 5 N activity had a good inverse correlation with blood lead concentration (r = -0 84).
.0
20 r=-o 84
-r
15 10 E z
to a,
5 0 0
20 40 60 80 Blood lead, pg/dl Fig 1 Correlation between erythrocyte pyrimidine 5'-nucleotidase (P 5N) activity and blood lead concentration in 22 lead workers
A
nl,
I
0 g~
+ 4C
H
U
1 a
I -" SLL 0
**
lU
L
a
50
cu
.I
min
B Fig 2A, B Chromatograms of erythrocyte nucleotides obtained with the HPLC method: A normal control; B lead worker CDPE, cytidine 5 '-diphosphate-ethanolamine; NAD, nicotinamide adenine dinucleotide; CDPC, cytidine 5 '-diphosphate-choline; CMP, cytidine 5 '-monophosphate; AMP, adenosine 5 '-monophosphate; IMP, inosine 5 '-monophosphate; UDPG, uridine 5'-diphosphate-glucose; NADP, nicotinamide adenine dinucleotide phosphate; ADP, adenosine 5 '-diphosphate; GDP, guanosine 5 '-diphosphate; UTP, uridine 5 '-triphosphate; CTP, cytidine 5 '-triphosphate; ATP, adenosine 5 '-triphosphate; GT', guanosine 5 'triphosphate
aD
o
U^
e a
Lw '9
min
421 Table 1 Correlation coefficient between nucleotide concentration and pyrimidine 5'-nucleotidase (P 5N) activity or blood lead concentration (Pb-B) in 22 lead workers Nucleotide
P 5N
Pb-B
CDPE NAD CDPC + CMP AMP IMP UDPG NADP ADP GDP UTP CTP ATP GTP
-0 09 -0 15 -0 54** -0 03 0 14 -0 42 * -0 06 0 08 0 07 -0 56** -0 54** 0 12 0 24
-0 01 0 34 0 49 * -0 01 -0 38 0 31 -0 04 0 02 -0 11 0 62** 0 62 ** 0 02 -0 29
Significance: *P
InternationalArchivesof
Oc Upationald
Environmental Itealth © Springer-Verlag 1992
Erythrocyte nucleotides in lead workers Masayoshi Ichiba l , Katsumaro Tomokuni', and Koji Mori 2
'Department 2
of Community Health Science, Saga Medical School, Nabeshima, Saga 849, Japan Nishinihon Occupational Health Science, Yahatahigashi, Kitakyusyu 805, Japan
Received June 25 / Accepted October 10, 1991
Summary We determined erythrocyte nucleotides levels in 22 lead workers with blood lead ranging from 8 to 78 jgldl Their erythrocyte pyrimidine 5'-nucleotidase (P 5N) activity ranged from 19 4 to 3 4z mol uridine/h per g hemoglobin A significant elevation of erythrocyte pyrimidine nucleotides was found, and their contents correlated inversely with erythrocyte P 5 N activity and positively with blood lead concentration In particular, the contents of uridine triphosphate (UTP) and cytidine triphosphate (CTP) exhibited a high correlation with P5N activity (r = -0 56, -0 54) However, its concentration in lead workers was estimated to be much lower than that in hereditary P 5 N deficiency.
In this study we examined the accumulation of specific erythrocyte nucleotides in workers occupationally exposed to lead and compared the data obtained with P 5 N activity and blood lead concentration.
Key words: Lead
Erythrocyte P5 N assay and nucleotides separation The high-performance liquid chromatography (HPLC) system consisted of two LC-9A pumps, SPD-6 AV detector, CTO-6 A column oven, SIL6B autoinjector, SCL-6B system controller, and CR-4 A data processor, all from Shimadzu Co , Kyoto, Japan Erythrocyte P5N activity was determined according to the method of Tomokuni and Ichiba (1986) and expressed as gmol uridine/h per g hemoglobin (Hb). The extraction of nucleotides from erythrocytes was performed as follows: blood (1 ml) was deproteinized by adding 2 ml 0 6 N HC 10 4, and it was centrifuged at 1200 g for 5 min The supernatant (2 ml) obtained was neutralized with 2 ml 0 2 M K2CO 3 The nucleotides were separated and determined by a modification of the method of Angle et al (1982) The extracts (30 gl) were injected onto an anion exchange column (Partisil 5 SAX, 4 6 x 100 mm, 5 Itm particle size; Whatman Inc ) and a gradient elution system (buffer A: 7mMKH 2PO4, pH 3 9; buffer B: 250 mM KH2 PO 4, 500 m M KCI, pH 4 5) was used at a flow rate of 1 5 ml/min An isocratic period of 5 min followed by a 25-min concave gradient from O% to 100 % gave nucleotides separation within 40 min The wavelength was set at 270 nm, and column temperature was set at 50 °C Chromatographic peaks were identified by comparison with reference standards purchased from Sigma Chemical Co.
Nucleotide
Nucleotidase
Introduction Pyrimidine 5 '-nucleotidase (P 5 N, EC 3 1 3 5) specifically dephosphorylates pyrimidine nucleotides Pyrimidine nucleotides resulting from RNA degradation in maturing erythrocytes are normally rendered diffusible by P 5N (Valentine et al 1974) By this mechanism, the maturing erythrocytes retain purine nucleotides as a source of ATP. P 5N activity is a useful indicator of lead exposure (Paglia et al 1975 ; Angle and McIntire 1978 ; Buc and Kaplan 1978 ; Mohammed-Brahim et al 1985 ; Sakai and Ushio 1986 ; Ichiba et al 1987) It was reported that the depression of P 5 N activity results in the accumulation of pyrimidine nucleotides in erythrocytes in clinical studies (Paglia et al 1977 ; Angle et al 1982) and in rabbits treated with lead (Angle et al 1980 ; Swanson et al. 1980) However, there are few reports on workers with occupational exposure to lead (Ichiba and Tomokuni 1990 ; Sakai et al 1990). Offprint request to: M Ichiba
Subjects and methods Subjects Lead-exposed subjects were 22 workers (age 46 ± 11 years) occupationally exposed to lead They were employed in the secondary lead smelter factory Heparinized venous blood samples were collected from them The separation of erythrocytes from whole blood was accomplished by the method of Beutler et al. (1976).
Blood lead analysis The concentration of blood lead was analyzed by flameless atomic absorption spectrophotometry after 10-fold dilution of whole blood with 0 1 NHNO 3 containing 1% triton X-100. Statistical method Data analysis employed Pearson's correlation coefficient.
420 Figure 2 shows the typical chromatograms of erythrocyte nucleotides obtained from a control subject and a lead-exposed worker The peak for uridine triphosphate (UTP) and cytidine triphosphate (CTP), which was undetectable in control subjects, appeared on the lead worker's chromatogram. Table 1 shows the relationship between nucleotides concentration and P5 N activity or blood lead level A significant correlation was found between P 5N activity or blood lead level and the concentration of CTP, UTP or CDP-choline plus CMP The data demonstrated that the occupational exposure to lead elevates the contents of erythrocyte pyrimidine nucleotides, such as CTP, UTP, and CDP-choline plus CMP These results were similar to those reported by Sakai et al (1990) In addition, they reported that the levels of purine nucleotides (ATP, AMP) are affected by lead exposure However, we did not obtain a significant relationship between concentration of purine nucleotides in erythrocyte and lead exposure in the present study. Several investigators have indicated that abnormal accumulation of pyrimidine nucleotides in erythrocytes
Results and discussion Figure 1 shows the correlation between blood lead concentration and erythrocyte P 5 N activity obtained from 22 lead workers The P 5 N activity had a good inverse correlation with blood lead concentration (r = -0 84).
.0
20 r=-o 84
-r
15 10 E z
to a,
5 0 0
20 40 60 80 Blood lead, pg/dl Fig 1 Correlation between erythrocyte pyrimidine 5'-nucleotidase (P 5N) activity and blood lead concentration in 22 lead workers
A
nl,
I
0 g~
+ 4C
H
U
1 a
I -" SLL 0
**
lU
L
a
50
cu
.I
min
B Fig 2A, B Chromatograms of erythrocyte nucleotides obtained with the HPLC method: A normal control; B lead worker CDPE, cytidine 5 '-diphosphate-ethanolamine; NAD, nicotinamide adenine dinucleotide; CDPC, cytidine 5 '-diphosphate-choline; CMP, cytidine 5 '-monophosphate; AMP, adenosine 5 '-monophosphate; IMP, inosine 5 '-monophosphate; UDPG, uridine 5'-diphosphate-glucose; NADP, nicotinamide adenine dinucleotide phosphate; ADP, adenosine 5 '-diphosphate; GDP, guanosine 5 '-diphosphate; UTP, uridine 5 '-triphosphate; CTP, cytidine 5 '-triphosphate; ATP, adenosine 5 '-triphosphate; GT', guanosine 5 'triphosphate
aD
o
U^
e a
Lw '9
min
421 Table 1 Correlation coefficient between nucleotide concentration and pyrimidine 5'-nucleotidase (P 5N) activity or blood lead concentration (Pb-B) in 22 lead workers Nucleotide
P 5N
Pb-B
CDPE NAD CDPC + CMP AMP IMP UDPG NADP ADP GDP UTP CTP ATP GTP
-0 09 -0 15 -0 54** -0 03 0 14 -0 42 * -0 06 0 08 0 07 -0 56** -0 54** 0 12 0 24
-0 01 0 34 0 49 * -0 01 -0 38 0 31 -0 04 0 02 -0 11 0 62** 0 62 ** 0 02 -0 29
Significance: *P
