in a complex matrix such as whole blood has re- quired predigestion, extraction, or both (9, 10), or long drying and ashing times (11) to minimize background.
CLIN. CHEM.
25/11,
1915-1918
(1979)
Determinationof Manganesein Whole Bloodand Serum Patricia A. Pleba& and Karl H. Pearson2
We describe methods for determination of manganese in whole blood and serum with Zeeman-effect flameless atomic absorption spectroscopy. These analyses are performed on a twofold or fourfold dilution of the specimen in Triton X- 100, 1 gIL. No predigestion or extraction procedures are required. The method of standard additions was used for quantitation. Within-run coefficients of variation for whole-blood manganese were 7.0 and 5.5% for 2.29 and 5.67 tg/L, respectively. For determination of serum manganese, coefficients of variation were 10.3 and 5.3% for 0.97 and 3.01 tg/L, respectively. Manganese detection limits for the assays were 3.0 pg. Whole-blood manganese concentrations, determined for 60 subjects, yielded a mean (±SD) of 9.03 (±2.25) igIL. Mean serum manganese concentration, determined for 20 subjects, was 1.82 (±0.64) tg/L. No correlation was found between blood manganese concentrations and age, sex, or smoking status.
AddItIonalKeyphrases: flameless atomic absorption spectroscopy
normal values
occupational hazards
#{149}
sorption, light scattering, and wavelength-dependent radiation. Background correction is obtained by placing the graphite cuvette in the field of a 11-kG (1.1 T) permanent
magnet.
During
atomization,
The major absorption
route is the gastrointestinal
tract, with
absorption being intimately linked to iron absorption (4, 6). Manganese is mainly excreted in bile; only a small fraction is excreted in urine (3, 6). The majority of the manganese in whole blood is bound to heme in erythrocytes (7). A small fraction in the serum is bound to a f31-globulin (8). Increased amounts of manganese in whole blood have been reported in rheumatoid arthritis (4), iron-deficiency anemia (3,5), and healthy individuals exposed to manganese (2, 3).
Flameless atomic absorption spectroscopy is a sensitive technique for the quantitation of manganese. However, determination in a complex matrix such as whole blood has required predigestion, extraction, or both (9, 10), or long drying and ashing times (11) to minimize background absorption. Analyte-shifted Zeeman-effect flameless atomic absorption spectroscopy offers accurate and reproducible background correction during atomization for broad-band molecular ab-
magnetic field. Thus, the parallel radiation contains background absorption and analyte absorption, while the perpendicular radiation contains only background absorption. Background absorption is corrected for by electronically
subtracting absorbance.
the perpendicular absorbance from the parallel Correctable background absorbances, which are
somewhat dependent upon the element being analyzed, are generally up to 1.7 absorbance units. Koizumi and co-workers have described in detail the theory and instrumentation of
Zeeman-effect
Department
of Chemistry,
The
Cleveland
State
University,
1 Present address: Department of Chemical Sciences, Old Dominion University, Norfolk, VA 23508. 2Also with the Division of Laboratory Medicine, The Cleveland Clinic Foundation, Cleveland, OH 44106. Address correspondence to this author at Cleveland State University. Presented in part at the 30th Pittsburgh Conference on Analytical
Chemistry and Applied Spectroscopy, Abstract March, 1979. Received
Apr. 30, 1979; accepted
Aug. 9, 1979.
702, Cleveland,
OH,
flameless
atomic
spectroscopy (12). This method is ideally suited analysis of trace metals in biological matrixes.
absorption for direct
Materials and Methods Apparatus Analyses
were
atomic absorption
performed
on a Zeeman-effect
spectrometer,
fiameless
Model 170-70 (NSI Hitachi,
Mountain View, CA 94043) equipped with a R955 photomultiplier tube (Hamamatsu Corp., Middlesex, NJ 08846), sensitive to low ultraviolet wavelengths. We used a cup-type graphite cuvette (Naka Works, Hitachi Ltd., Katsuta Ibaraki, 312, Japan) for all analyses. Atomic absorption signals were
recorded on a Hitachi fast-response, dual-pen recorder, Model 056, which allowed simultaneous monitoring of analyte and background absorption. Thus, we could determine whether the background absorption remained below the correctable amount
of 1.7 absorbance
was regulated
units.
with an external
The flow of argon carrier
tubes (1.5-mL capacity,
Pittsburgh, PA 15219) and NY 14602) were soaked in tific, ACS reagent grade) distilled de-ionized water
gas
flow meter (Lab Crest Scien-
tific Glass Co., Warminster, PA 18974), which allowed flow to be adjusted from 0 to 100 mL/min.
All polyethylene Cleveland, OH 44115.
field produces
mically and hypsochromically, respectively. A rotating Senarmont quartz polarizer, placed between the hollow cathode lamp and the graphite cuvette, polarizes the hollow cathode lamp’s radiation alternately parallel and perpendicular to the
analyte-shifted Manganese is essential for bone and tissue formation, carbohydrate metabolism, reproductive processes, and lipid metabolism (1). Human manganese metabolism has been extensively studied with radioactive tracer techniques (2-5).
the magnetic
splitting of the electronic states of the analyte. The nonshifted, or ir, component, which is polarized parallel to the magnetic field, is located at the original transition wavelength; wavelengths of the o and (7+ components, which are polarized perpendicular to the magnetic field, are shifted bathochro-
the gas
Fisher Scientific,
volumetrics (Nalge Co., Rochester, nitric acid (6 mol/L; Fisher Scienovernight and then rinsed with before use.
Reagents Triton solutions,
X-100 (Rohm & Haas Co., Philadelphia, 1 gIL, were prepared with distilled
PA 19105) de-ionized
water. Stock manganese
standard
(1.000 gIL) was purchased
CLINICAL CHEMISTRY,
Vol. 25, No. 11, 1979
from
1915
8
Table 1. Zeeman Atomic AbsorptIon Spectroscopy Instrument Parameters for Manganese Analyses Dry:
Whole-blood analysis
Ramp mode, 0.3 A/s, 30 A (135 #{176}C)
S
Serum analysis
Ramp mode, 0.2 A/s, 30 A (135 #{176}C) Ash: Step mode, 60 s, 60 A (350-450 #{176}C) Atomize: Step mode, 8 s, 300 A (2400 #{176}C) Argon carrier gas flow: 50 mL/min Argon sheath gas flow: 3 L/min Wavelength: 279.5 nm Pen response: Fast (1) Band pass: 1.1 nm (slit 2) Lamp current: 10 mA Expansion: 0.1 absorbance unit/full scale
YLLLL
I-.
3
C
Fisher Scientific. For working manganese standard, we made two equal serial dilutions of the stock standard with distilled de-ionized water to give a final manganese concentration of 100 igIL.
Collection
and Preparation
E
of Samples
All venous blood specimens were collected in Trace-Element Vacutainer Tubes (Becton-Dickinson, Rutherford, NJ 07071). Whole-blood specimens were drawn in Vacutainer Tubes with sodium heparmn additive, and serum specimens were collected in silicone-coated Vacutainer Tubes with no additive. Whole-blood specimens were stored at 4 #{176}C and assayed within four days. Serum specimens were allowed to clot and then centrifuged at 3500 X g for 30 mm. All hemolyzed specimens were transferred Serum days.
specimens
were discarded. The remaining specimens to acid-washed 1.5-mL polyethylene tubes. were stored at 4#{176}C and assayed within four
Procedure For whole-blood
specimens,
we transferred
with
a posi-
tive-displacement pipette (Scientific Manufacturing Industries, Emeryville CA 94603) 0.25 mL of the well-mixed specimen to an acid-washed polyethylene tube. We added 0.75 mL of Triton X-100 solution and mixed by inversion. A standard curve
was prepared
making standard manganese
from
additions
diluted
whole-blood
and subtracting
concentration
matrix
by
the endogenous
to give 0, 2.5, 5.0, and
10.0 tg of
manganese per liter. We then transferred a 10-FL aliquot of the diluted specimen or standard with a variable automatic pipette (Excalibur Laboratories, Ltd., Australia) to the graphite 1.
cup. Analytical
Average absorbances
conditions
of duplicate
were as shown
pipettings
in Table
(triplicate
if
duplicates
differed by more than 10%) were compared with the standard curve. We obtained whole-blood manganese concentrations by multiplying by 4 the concentration read from the standard curve. For serum manganese concentrations, we diluted 0.5 mL of the specimen with 0.5 mL of Triton X-100 solution and
transferred
a 15-zL aliquot
to the graphite
cup cuvette.
multiplied the concentration of the diluted specimen by 2. Figure 1 shows a recorder trace of a serum manganese standard curve. A plot of these data yields a line with equation y = 13.lx + 7.4 and a goodness of fit of 0.9999. The abeorbance CLINICAL CHEMISTRY,
Vol. 25, No. 11, 1979
Fig. 1. Serum manganese standard curve A, endogenous manganese conce*atlon (0.6 ig/L);B, endogenous+ 2.5ig/L; C,endogenous+ 5.Oig1L; D,endogenous+ 10.Og/L; E,specimen(1.OigIL). Bottom trace: full scale = 0.1 absorbance unit Top trace Is background absorbance (see ref. 13). Background peaks occur during ash cycle, F, and atomizatlon cycle, 6
during the ash cycle, F, is due to smoke formation, and the atomization cycle, G, is total absorbance. The maximum background absorbance during atomization with an initial photomultiplier voltage of 340 V was calculated as 1.2 absorbance units (13), well below the maximum correctable absorbance
of 1.7.
Results and DIscussion We evaluated the Trace-Element Vacutainer Tubes for manganese contamination. Distilled de-ionized water was drawn into 10 Vacutainer Tubes through Becton-Dickinson disposable needles. No detectable manganese was found in the sodium heparmn Vacutainer Tubes, and only two Vacutamer Tubes with no additive showed traces of manganese (0.25 and 0.36 ig/L). Next, we used whole blood to study leaching in the heparinized Vacutainer Tubes. Four specimens were analyzed for manganese immediately upon collection and again five days later. There were no significant changes in the manganese concentrations for these specimens. The detection limit for manganese by this method is 3.0 pg, as calculated from 2SD at a concentration near the detection
In-
parameters were the same as those for whole-blood except for the drying cycle. A matched serum-matrix standard curve was also prepared in the same manner as for wholeblood determinations. We corrected whole-blood and serum sample absorbances for a reagent blank before comparison with the curve. For serum manganese concentrations, we strument
1916
LL
Table 2. WithIn-Run Precision StudIes CV%
Whole-blood analysis (n
=
2.29±0.16 5.67 ± 0.31 Serum analysis (n
0.97 ± 0.10 3.01 ± 0.16
20)
7.0 5.5 =
20)
10.3% 5.3%
Table 3. Average Normal Blood Manganese ConcentratIons n
i±SD,Mg/L
Zeeman-effect atomic absorption 60 spectroscopy Neutron activation analysis 14 14 limit. At 2.29 tg/L pg for a 10-L
the detection
aliquot.
This
Table 4. Effect of Sex and SmokIng Status on Blood Manganese n
Ref.
9.03 ± 2.25 9.84 ± 0.4 8.44 ± 2.73
4
15
limit is 0.3 ig/L, detection
which is 3.0 is adequate to in whole blood and to limit
detect human manganese deficiency quantitate normal values for manganese in serum. The standard curve for the assay is linear to 10 igIL in the diluted specimens, which allows quantitation of increased manganese concentrations without further dilution because whole-blood manganese concentrations with up to 80 igfL can be analyzed from a 5-jL aliquot. The linearity range of the standard curve drops rapidly with decrease in carrier-gas flow. Table 2 shows results of within-run precision studies for the assay of manganese
and in serum. To determine between-run precision of whole-blood ganese assay, we made 44 replicate determinations
specimens
in whole blood
over a period
of eight days. The coefficient
of
variation variability
(CV) for the assay is a measure of the expected in sampling between patients as well as day-to-day variation (14). The standard deviation was 1.04 igfL at a mean concentration of 10.5 tg/L, giving a CV of 9.9%. For the
serum manganese
assay, 28 replicate
determinations
eight specimens
26
9.22±
34
Nonsmokers
31
8.87 ± 2.35 9.24 ± 2.33
Smokers
29
8.87±2.19
tration
made on
(most of which were in the 1.0-2.0 igIL range) had a mean concentration of 1.69 tgIL and standard deviation of 0.22 ig/L, giving a CV of 13.0%. Whole-blood specimens obtained from 60 nonfasting, apparently healthy volunteers (26 men and 34 women), ranging in age from 15 to 83 years, had a mean manganese concen-
of 9.03 ± 2.25 zg/L
compares
(range,
well with published
3.85-15.1
2.15
Lg/L).
This
results for neutron
activation 3. By Student’s
analysis (4, 15). Results are shown in Table t- test, these analyses are not significantly different.
We found
no correlation between age and whole-blood manganese concentrations (Figure 2), and no correlation between the sex of the subject or smoking status and whole-blood manganese concentrations
(Table
4).
Mean serum manganese concentrations for 20 nonfasting, apparently healthy individuals (six men and 14 women), ranging in age from 18 to 64 years, (range, 0.94-2.92 igfL).
Table 5 summarizes
manon 19
±SD,Mg/I.
Men Women
and those of several
analysis
the comparison
investigations
and graphite
were used (2, 4, 15-17).
were
furnace There
1.82 ± 0.64 zg/L
between
in which neutron
flameless
atomic
is a wide range
our results activation
absorption
of results
even
between neutron activation analyses. Whether this reflects external contamination or a method difference cannot be ascertained from the results. The neutron activation procedures all require chemical separation of manganese from the whole-blood or serum matrix, which could result in analyte losses and account for the lower values. We feel that the assay for whole-blood manganese will be useful in investigating manganese nutritional status as well as monitoring manganese exposure in the general population. Mena
et al. (3) indicate
that
healthy
individuals
with occu-
15 14 13
12 11 Mn Conc, 10
------.--.-
-
-.‘-_...__#{149}____
-
--
_..!
-:=
L
8
9.03
1SD
7 6 5 4
3
2 1
10
20
30
40 50 Age, years
60
70
80
Fig. 2. Relation of whole-blood manganese concentrations to age of donor CLINICALCHEMISTRY,Vol. 25, No.
11, 1979
1917
Table 5. ComparIson of Serum Manganese Determinations n
Ref.
Zeeman-effectatomicabsorption 20 spectroscopy
1.82± 0.64
Flameless atomic absorption
1.91
50
17
spectroscopy 15 14 14 50
analysis
1.29±0.11 1.42±0.2 0.59±0.18 0.57±0.13
2 15 16
pational exposure to manganese exhibit significantly increased values for whole-blood manganese. Use of neutron activation analysis for whole-blood manganese determination is expensive and time-consuming. Zeeman-effect flameless atomic absorption spectroscopy allows quantitation of manganese in whole blood and serum with results comparable to those by other analytical techniques.
gratefully
acknowledges
a CSU-RIA
research
References 1. Leach, R. M., and Lilburn, M. S., Manganese metabolism and its World Rev. Nutr. Diet. 32, 123 (1978). 2. Cotzias, G. C., Horiuchi, K., Fuenzalida, S., and Mena, I., Chronic manganese poisoning. Clearance of tissue manganese concentrations with persistence of the neurological picture. Neurology 18, 376 functions.
(1968). 3. Mena, I., Horiuchi, K., Burke, K., and Cotzias, G. C., Chronic manganese poisoning. Individual susceptibility and absorption of iron. Neurology 19, 1000 (1969). 4. Cotzias, G. C., Papavasiliou, P. S., Hughes, E. R., et al., Slow
1918
(1966). Borg, D. C., and Cotzias, G. C., Incorporation
7.
Neutron activation
Karl H. Pearson grant.
turnover of manganese in active rheumatoid arthritis accelerated by prednisone. J. Clin. Invest. 47, 992 (1968). 5. Mahoney, J. P., and Small, W. J., Studies on manganese. III. The biological half-life of radiomanganese in man and factors which affect this half-life. J. Gun. Invest. 47,643 (1968). 6. Bertinchamps, A. J., Miller, S. T., and Cotzias, G. C., Interdependence of routes excreting manganese. Am. J. Physiol. 211, 217
CLINICAL CHEMISTRY,
Vol. 25, No. 11, 1979
of manganese
into
erythrocytes as evidence for a manganese porphyrin in man. Nature 182, 1677 (1958). S. Foradori, A. C., Bertinchamps, A., Builbon, J. M., and Cotzias, G. C., The discrimination between magnesium and manganese by serum proteins. J. Gen. Physiol. 50, 2255 (1967). 9. Suzuki, M., and Wacker, W., Determination of manganese in biological materials by atomic absorption spectroscopy. Anal. Biochem. 57,605 (1974). 10. Buchet, J. P., Lauwerys, R., and Roels, H., Determination of manganese in blood and urine by flaineless atomic absorption spectrophotometry. Clin. Chim. Acta 73, 481 (1976). 11. Bek, F., Janouskova, J., and Moldan, B., Determination ganese and strontium in blood serum using the Perkin-Elmer graphite furnace. At. Absorpt. Newsl. 13,47 (1974).
of manHGA-70
12. Koizumi, H., Yasuda, K., and Katayama, M., Atomic absorption spe#{233}trophotometrybased on the polarization Zeeman effect. Anal. Chem. 49, 1106 (1977). 13. Analytic Spectrometer, 1-5.
Techniques for the Zeeman Model 170-70. Hitachi
Effect
characteristics Atomic
of the
Absorption
Ltd., 1978, Appendix 1, p
14. Bauer, E. L., A Statistical Manualfor Chemists. Academic Press, New York, NY, 10003, 1971. 15. Cotzias, G. C., Miller, S. T., and Edwards, J., Neutron activation analysis: The stability of manganese concentrations in human blood and serum. J. Lab. Clin. Med. 67,836 (1966). 16. Versieck, J., Barbier, F., Speecke, A., and Hoste, J., Normal manganese concentrations in human serum. Acta End ocri not. 76, 783 (1974).
17. Stevens, R. J., Pradhan, N. K., Atkins, R. C., and Thompson, N. P., Preliminary studies on trace metal changes in blood during haemodialysis. In Clinical Chemistry and Chemical Toxicology of Metals, S. S. Brown, Ed., Elsevier/North Holand, New York, NY, 1977, p 45.
