The determination of zinc in blood plasma by atomic absorption spectrometry

Analytica Chimica Acta, 94 (1977) 63-73 0 Elsevier Scientific Publishing Company,

Amsterdam

-Printed

in The Netherlands

THE DETERMINATION OF ZINC IN BLOOD PLASMA BY ATOMIC ABSOR.PTION SPECTROMETRY 0

GERALD

Department WILLIAM

P. BUTRIMOVITZ

of Chemisty.

University

Department of Chemistry, JfcGill Quebec. H3A 2K6 (Canada) (Received

of Maryland,

College Park, hlD 20742

(U.S.A.)

C. PURDY*

26th April

University.

801 Sherbrooke

Street

West, Montreal,

1977)

Atomic absorption spectrometry has been used in the analysis of plasma zinc because of its sensitivity and simplicity. Dilution techniques reduce the viscosity of plasma and facilitate direct analysis, but viscosity differences can produce deviations in aspiration rates between sample and standard, and so cause errors. A direct (1 + 4) dilution of plasma with deionized mater is suggested. Working zinc standards are prepared in 5% glycerol to approximate the viscosity characteristics and aspiration rates of the diluted plasma samples. The analytical cumes for diluted plasma samples and 5% glycerol working standards proved identical. Plasma zinc concentrations are accurately calculated from a daily working curve. The accuracy of the method exceeds 99% and recovery of added inorganic zinc to a pooled plasma averages 99.8%. The precision is primarily limited by baseline drift. A confidence interval of 2 2 pg/lOO ml was achieved by means of six contiguous 10s~integration readings. The method is free of nebulizer clogging and matrix interferences and is uot subject to significant day-today variations. Because the method is accurate, sensitive, reliable and specific. it should be useful in the clinical laboratory_

Plasma zinc has been assayed by various procedures including calorimetry, neutron activation, fluorimetry and atomic absorption spectrometry (ULS.) f&2]. A.a.s. has been used for most routine analysis because of its low cost of operation, sensitivity and precision [2,3]. Even in the presence of a complex ionic matrix, analgsis for zinc can be interference-free (41. Before the advent of the Baling burner, which is suited to high solids matrices, sample preparation required precipitation of the plasma proteins by trichloroand costly, and trace conacetic acid [5] . The method was time-consuming taminants could be introduced 161. Sprague and SIavin [7] suggested a direct method for plasma zinc requiring a (1. + 1) dilution with water. The zinc standards were also prepared in water. Although the method was free of chemical interferences, the burner head had to be modified to prevent clogging 8 Taken iu part from b¶ary!and, 1977.

the PhD.

dissertation

of Gerald

P. Butrimovitz.

University

of

from plasma prot&.ns. Accuracy was poor, perhaPs because of flow rate or visco.sity differences between sample and standard. Hackley et al. [8] also used a (1 + 1) dilution with water; the zinc working standards were prepared in 3Yc dextran solution to match the viscosity of the diluted plasma and recoveries averaged 99%. Reinhold et al. [9] further explored the effect of various flow r&es and viscosities on plasma zinc analysis. They pointed out tl iat when dilute plasma samples were aspirated through thin capillaries, significant changes occurred in flow rate and absorbance readings, because of apparent viscosity differences between plasma sample and standard. They concluded that a minimum twofold dilution was necessary for precise and accurate meesurements. In order to obviate errors from clogging, others [10-131 suggested X0and 2O-fold dilutions, but a lowered signal sensitivity necessitated the addition of nitric acid or n-butanol as enhancing agents [lo, 111. Extreme dilution may also contribute to reduced precision from greater pipettbrg errors, inhomogeneity of the dilute sample and a relatively low signal-to-noise ratio [lo] . Because the viscosity effect of dilute plasma cannot be totally reduced by extreme dilution, Pekarek et al. [ 131 suggested a method utilizing (1 + 4) dilution of plasma, which gave higher signal sensitivities without capillary clogging. Mthough reproducible values were obtained with aqueous s-tidards, the accuracy of the values was not confirmed. It has been suggested [ 141 that the largest source of error in flame methods arises from differing viscosities and thus flow rates between sample and standard. Glycerol has been previously used as a viscosity adjuster for clinical measurements [ 131. Itsversatility comes from its capacity to maintain homogeneity with buffer solutions over a wide range of concentrations. Its flow and viscosity characteristics are well defined [la, 153 . An aa.s. methods handbeok [IS] suggests that the viscosity of a (1 + 4) diluted serum sample nearly matches the viscosity of zinc standards prepared in 5% glycerol. In the method described below, the one-step dilution of plasma with a fourfold addition of deionized water is recommended. Working zinc standards are prepared in ~Yo glycerol solutions to approximate the viscosity and aspiration rates of the diluted plasma samples [9,14,16] . Because the technique is accurate, sensitive, reliable and specific, it !L suitable for routine clinical laboratories [ 173. EXPERIMENTAL

Reagents and materials All reagents, dillrents and containers utilized were regai&d as possible sources of zinc contamination and were subjected to a continuous quality control program. Although sources of materials are given, other sources may prove satisfactory. All volumetric glassware must meet NBS Class A specifications. Glasswsre and Pasteur pipets are acid-washed (No-Chromix, Godax Labcra~ries,

65

New York, N.Y. 10013),soakedin 1% Na?EDTA solution for 24 h and rinsed 6 times with deionized water. Disposable serological pipets 0.5 ml (Borosilicate glass; Nimble-Division, Owens-Illinois, Pittston, Pa. 18640) and polystyrene tubes (16 X 25 mm; Falcon Division, Be&on, Dickinson and Co., Oxhard, California 93030) were routinely found not to contribute detectable concentrations of zinc and required no pretreatment. Glycerin (Certified A.C.S., 99.4% Fisher Scientific Comp’any, Silver Spring, Maryland 20910) was used to prepare a 5% (v/v) glycerol solution in deionized water. The deionized water had a specific resistance of at least 10” ohms at 25’C. Stock zinc standards. For the primary zinc standard (1000-ppm zinc), dissolve 1.000 g of zinc metal (Zinc Powder-200 mesh, Alfa Inorganics, Beverly, Mass.) in 50 ml of dilute (If 4) nitric acid (Ultres Nitric Acid, J.T. Baker Chemical Co.) and further dilute to 11. The secondary standard (lOOO-ppm zinc) was Fisher Scientific Certified Standard. Working standards are prepared as follows. Deliver 1 ml of lOOO-ppm zinc standard to a lOO-ml volumetric flask and dilute with 5% $ycerol to produce a 10-ppm zincs% glycerol solution. Invert the solution 16 times. Add 1,2, 3 and 4-ml aliquots of this solution to loo-ml volumetric flasks and dilute with 5% giycerol to produce 10,20,30 and 40 pg/lOO ml zinc working standards_ Invert the solutions 16 times. Plot they standards on the working ctuve as apparent concentrations of 50,100,150,200 pg/lOO ml, because plasma samples are diluted to one-fifth their original concentration_ The zinc concentration of the plasma samples are thus read directly from the working curve. Prepare standards daily. Pooled plasma standards. P&ma (e.g., obtained from plasma packs) is pooled, centrifuged at 900 g for 20 min and decanted. Aliquots are stored in polyethylene vials (which.were found to contribute no zinc) at -20°C and are stable for at least one year. Collection

and handling of specimens

Collect 3-5 ml of blood by venipuncture with all plastic polyethylene syringes (Peel A Way Scientific, So. El Monte, California 91733) and stainless steel needles (Monojet+250. Sherwood Medical Industries, Inc., Delano, Florida 32720) or Butterfly (21 or 23) infusion sets (Abbott Laboratories, North Chicago, Ill., 60064). Add 2 drops (0.10 ml) of a 30% (w/v) sodium citrate (reagent grade) anticoaggant solution to the syringe before blood collection. Centrifuge promptly at 900 g for 20 min with the syringe serving as the centrifuge tube.Pipette the plasma into polyethylene vials (MiniScintillation Vials; Fisher Scientific, Cat. No. 3-337-20) and freeze at -20°C. Care is taken not to disrupt the buffy coat or packed cells. Hemolyzed samples are discarded; platelets and red cells introduce high levels of zinc into the sample [ 18, 191.

Procedure Prepare working standards as described abose. Thaw plasma samples at room temperature and invert 6 .times. Transfer a 0.5ml sample with a serological pipet w a~l25-mm test tube. Add 2 ml of deionized water. Cover the tube with a parafilm slip and vortex for 30 s. Repeat this for plasma samples in groups of 10. Prepare a pooled plasma sample similarly. Establish instrumental and gas flow settings precisely as listed in Table 1. Once the aspiration rate is established for IO-ml samples of water, lock the nebulizer flow adjustment in place; to facilitate the m-establishment of the spiration rate, a groove may be etched onto the assembly. Aspirate a 5% glycerol solution and establish the baseline in the absorbance mode to read 0.000 + 0.001. A baseline reading is taken before and after each sample and m-established as required. Aspirate the zinc working standards sequentially, from mo.% dtite to most concentrated, until stable readings (+O.OOl) are achieved and record the following six contiguous lo-s-integration absorbance readings. Three readings produce slightly inferior precision. Take the average for each standard. Use these values to establish the daily working curve, preferably by a regression least-squares fit. Vortex a pooled plasma sample again and aspirate. Calculate the concentration by interpolation from the working CuNe. Results must be within 2 pg/lOO ml of the established value. (Th0ugh.a plasma zinc standard is not yet available, a laboratory reference standard can be established by the method of additions [20] . The precision of this method may f7uctuati with the condition of the equipment.) Finally voptex plasma samples and read similarly in groups of ten. Aspirate working standards after each group.

Instrumental settings for the Perkin-Elmer 403 atomic absorption with Soling burner head and zinc intensitron lamp

Imtrumrntal

settings

Wavelength Sit width Mode Lamp Current

213.8 nm 4 AbsorbancelOs 15 mA

integration

Gain Lamp focus Burner height Flame

Gas flow settings Pressur~(psi) Air Acetylene

Aspiration tBumcr

30: 9

note (H,O)

FIowmeter 54 ,38

5.0 + 0.1 ml min*

height is adjusted just to intersect the lamp beam.

_

spectrophotometer

Midscale Grazing burner head’ 7.75a Luminescent (fikl-rich)

67 RESULTS

AND DISCUSSION

Standard working curve Figure 1 illustrates a typical working curve for zinc standards prepared in 5% glycerol_ Several plasma samples of known zinc concentration (Table 5) are also plotted. The analytical curve formed by these plasma samples is identical with the curve for the working standards (Table 2). Thus the plasma matrix at a fourfold dilution has no marked effect on zinc measurements within the physiological range. The linearity of these cumes was established to 200 ng/lOO ml. Effects of varyingglycerol concentrations When zinc standards are prepared in 0 and 10% glycerol solutions, the resulting curves (Fig. 2) are significantly different than either the zinc--5% glycerol or plasma zinc curves (P < 0.05, Table 2). The multiplicative errors that would arise in estimating a pooled plasma from these standard curves are reported in Table 3. Though the errors appear slight, they are significant. The aspiration rates for these solutions axe &so presen&l, illustrating the well-known fact that the population of zinc atoms measured in the flame is regulated in part by the velocity of sample aspiration, which is controlled by the viscosity of the solution [ 141; 98-99% of the viscosity of the plasma is accounted for by the protein concentration [ 223 . Viscosity measurements were carried out on O%, 5%, lo%, glycerol and diluted plasma samples with a capillary Cannon-Master viscometer 1231 at 25°C (Table 4). Clearly, the viscosity of a 5% glycerol solution approximates that of a diluted pooled plasma. Harkness 122 ] presented a viscosity (cP)-total protein (T.P.) plot for normal plasma solutions. By extrapolating this semi-log plot to 0 g% T.P., theoretical viscosity values were caIculated for dilute plasma solutions (Table 4). .A fourfold dilution of the pooled pl_asma (T.P. = 6.2 g%) produced TABLE

2

t-Test for two slope9

Slopes test&b

t Value

Significance

5-O% glycerol 5-10% g!ycerol O-1090 glycerol Plasna-5% gIycerol Plma-0% giycerol PlesmhlO% glycerol

t = 4.01 t = 3.54 t = 7.58 t= 1.23 t = 2.37 t -.4.39

P < P < P < NS. P < P<

bFias 1 and 2.

between

0.01 0.05 0.01 0.05 0.01

(1 sided)

slopes

66 ICOr

so

I

r

Zinc(pq/lO@

Zinc(jtq/lOOml)

Ial)

Fig_ 1. Standard working curve for zinc in 5% glycerol (9). Plasma samples of known zinc concentration (_* 2 s.d_) are aIso piottt (o)_ Fig. 2. Standard working curves for zinc. o 0% glycerol.

0 5% glycerol.

8 10% glycerol.

a solution with a protein content of 1.24 g%. Calculation of viscosity from this relationship yielded-a theoretical value of 0.98 cP, identical to that found experimentally. arther validity of these measurements was established thnzqgh the viscosity measurement of water. ‘A value of 0.90 cP was found, in agreement wiih the literature value of 0.89 cP. TABLE

3

Errors arising from tbe use of different standard curves. Matrix

Relative concertration readings (fig/100 ml)= Aspiration rate, (ml min*) Viscosity (c-“): experimentalb theoretical= Flow (ml min-) ‘Woncentrations working curare.

0%

5%

10%

Pooled

glycerol

glycerol

glycerol

Pha

84.1

91.7

95.3

91.9

6.0 0.90 0.89 2.00

5.7 1.04

5.4 1.25

1.76

1.50

were calculated from an absorb&e

of 0.038

bMeasurements of viscosity and flow were taken at 25.0% with flow times exceeding 125 s. ‘Xdculated from reported data [22]-

5.8 0.98 0.98 1.84 with respect to each

with a Cannon-Master

viscometer

69 TABLE

4

Viscosity Viscomete+

measurements

mlb

Mat&X

ta” 6)

FIOW
Kinematic ViSCOSitY t, x c

dad tsm1-Q

Absolute viscosity 'labs

Ml04 M 104

water 55i.glyceml

3.64 3.64

108.94 124.39

2.00 1.76

0.89993 1.02756

0.997 1.011

0.897 1.039

Ml05 NlO.4

lO%glycerol mamad

3.54 3.64

141.20 118.52

1.50 1.84

1.21856 0.97907

1.025 1.002

1.249 0.981

Caleolcted

Viscositye ME protein) 1.2& 1.32-l.6Sf

0.98

0.98-1.02 0.93

a7s

aCannon-3%3ster Viscometer at 25°C. bVolume of aqueous charge in viscometer. c Density measured with a Hubbsrd Pycnometer at 25OC. d’Jalue represents a fourfold dilution of a pooied plasma (6.2 g%)_ eDat.a approximated from published dati [22]. ‘Values represent a fourfoId dilution of normal plasmas(6.6-8.4 gS).g Value represents a ninefold dilution of a typical plasx~~(7.5 gS).

Normal plasma protein concentration may range from 6.6 to 8.4 g%. Through fourfold dilution, the otherwise wide viscosity range is minimized. The contribution of proteins to the viscosity of ninefold diluted plasma is still substantial (Table 4). It appears that aqueous zinc solutions are inappropriate as working standards in the measurement of diluted plasma The similar zinc concentrations, aspiration rates, viscosities, and fiow rates for the 5% glycerol standard and diluted pooled plasma sample (Table 3) confirm the need for adjusting the aspiration rate of the standard to the sample.

Standard

addition

curve

The method of standard additions [Zl] was utilized to establish the zinc concentration of a plasma pool as 91.9 pg/lQO ml. The concentration of the plasma pool was calculated from the 5% glycerol working curve to be 91.7 ug/lOO ml. The essential identity of these results indicates that there are no chemical interferences in the determination of zinc in fourfold diluted plasma. Recovery study Accuracy was further tested through recovery studies in which varying concentrations of inorganic zinc(II) was added to a pooled plasma. Recovery was calcuk&ed from the zinc-5% glycerol working curve (Table 5); the average recovery was 99.8%_ Plasma zinc concentrations could therefore be calculated accurately from the zinc-5% glycerol working curve. Newer a.a.s. instruments are equipped with direct concentration readouts. Once linearity of the working curve has been established, the instrument is calibrated wi’th a single zinc standard. In a typical series of analyses (6 separate runs) of a pooled plasma sample by the factor method, the average zinc recovery was 99.2% ( f 2.1% s-d.), when a 20 pg Zn/lOO ml--5%

70 TmLE

5

Recovar~ of zinc added to pooled Zinc added (JJg/100 ml) -0

Found (Pg1100

25 50 75

114 143 169

plasma Expected __ (rg/100 ml)

mi)

92

Recovery (%)

-

-

117 142 167

97.4 100.7 101.2 d = 99.8

_, 2.1

glycerol working standard was set to read 100 c(g/lOO ml; for this series, each of the six values was the average of six~successive readings on each sample. Although a daily working curve is required to insure accuracy, reasonable accuracy may be achieved through duplicate preparation and determination of a working standard. Precision -4 study based on a hierarchical design 1211 was undertaken to evaluate the contribution cf components creating variance (Table 6). The components evduated were arranged in tiers as “szimples” to account for sampling discrepancies, as “dete.rminations”-to account for drifts in the baseline perhaps caused by gas and sample flow fluctuations and inhomogeneity of the sample, TABLE

6

Precision (hierarchical design)a Samples D&enminations

Readings

A --a

b

a

b

a

b

38 39 39 40

-39 39 38 40

39 41 39 40 40 40

41 40 39 41 41 40

39 39 40 40 41 40

39 38 38 37 38 39

B

3”i: 2 = 39.4,

s-d. = 5 1.0, &a.

c

= 2.5%

Anaiysis of variance F=(d

-samples e te rminations within samples)

= 1.42

,.= (determinations Error

within

within &rnples) = 5.50 dete!rmination

2Value5 are given & Absorbance

X 1000

.(2,3 (3;3d

6F) DF)

N.F.’ 9 < 0.01

71

and as “readings” to account for instrumental noise. 4 plasma pool was sampled in triplicate and each sample determined in duplicate. A mean absorbance reading of 0.0394 was calculated with a standard deviation of ~0.0010. The results of the analysis of variance indicated that (a) the sampling procedure did not contribute to the variance (P > O-05), and (b) that significant variance (P < 0.01 j was introduced from within the series of determinations The variance from within the series of determinations might be caused by baseline drift. This further suggests a need for vortexing the sample and establishing the zero baseline prior to each sampling period (see Procedure). Duphcati determinations will obviously increase the precision of the method. Confidence intervals [ 211 were calculated from these data based on one, three, and six readings and converted into concentration intervals (Table 7); accordingly, six readings are suggested to achieve high precision, although the use of three readings results in only a minor loss of precision.

Interday

variation

The use of a daily working curve reduces the effects of interday instrumental and gas fluctuations [20] _ An interday variation study was therefore undertaken. Thirty-four samples representing a random selection from eighty previously analyzed samples were reanalyzed (Table 8). The r-test for paired samples 1211 showed no significant difference (P > 0.05) between the two sets of data. The results further illustrate that samples can be stored at -2O”C, thawed, and accurxtely reanalyzed.

Ion interference High salt concentrations interfere with the accurate determination of zinc in water [5], but the degree of intiference varies with the salt concentration [7]. The effect of interfering ions at physiological levels can be limited by dilution techniques. Although plasma ion concentrations remain rather constant in the adult, they may increase as a result of disease [24]. To evatuate the limit of accuracy of this method in elevated states, major ions reported to interfere [73 were added to a plasma pool (Table 9). The concentrations added were equal to eight times the standard deviation of normal ion TABLE

7

Confidence

intema!s

For a single reading For three readings For s-k readings

Absorbance (X 1000)

Canccntrntion

39.4 t 2.0 39.4 *- 1.2 39.4 2 0.8

88.7 L 4.5 88.7 + 2.7 88.7 5 1.8

(rgi100

ml)

72 TABLE

8

Interday variation

10/75 G/75 10175

63 63 53

75 78 50

68 70 65

60 60 78

75 78 55

6175

50

50

63

75

55

t-test (paired observations)

t= 0.63

c>

90 95

48 55

85 85

80 80

70 75

55 55

53 70 60 ‘70

6G 68

80 68

73 73

80 85.

80 83

65 68

70 68

65 75

75 65

55 53

95 95

95 90

80 70

65 58

85 85

70 63

(N=-,34)

0.05

N.S.

Mean difference (X) = G-529 S’ar.dard error (SX) = 4.876

plasma concentrations [24]. The plasma pool was sampled in triplicate and each sample was determined in duplicate; analysis of variance indicated that there is no significant effect of these ions on plasma zinc determinations (P> 0.05).

An acc*urate,sensitive and specific method for the determination of zinc in human plasma by aas. through a fourfold dilution with water has been studied. Sixnilar analytical curves, aspiration rates and viscosities were achieved with pooled plasma and zinc+% glycerol working standards. SigCficant errors were incurred when water or zinc-_1096 glycerol wcrking standards were utilized. The concentrations of zinc in a plasma pool calculated from an additions curve and from a zinc-W6 glycerol working curve were identical. Recovery of plasma zinc was 99.2% when calculated from a single standard. The precision of the method was evaluated by a hierarchical design. The standard deviation was +O.OOIO and the confidence interval was ?0.0008 for a series of six contiguous lo-s-integration absorbance readings. The concenTABLE

9

Effect of ions added to pooled plasma Ion (salt)”

bfeq Added

Absorbance

K*(KCl) Na’(NaC1) H,PO,-(KH;PO,j H,PO,- (NaH$O,)

4.0 30.0 3.2 3.2

37.9 37.4 37.8 37.6 37.3

-

-

Ana!ysis of variance k A 0.687; .

P > O.OS;dF;

.x 1000

t-paired

t = l.&.N_S. dF-4b

> = 4=; dFz = 25.d

.. aReagent grade. bDegrees of freedom -. total. =Degrees of freedom of freedom - measurements.

-

samples. &Degrwza

73

tration confidence interval was *1.8

pg/lOO ml for six readings. The significance of thevariables contributing to the precision of the method was evaluated

by analysis of variance for a hierarchical design. Basetine drift was a major cause of the variance, whereas sampiing errors were not. Effects from daytoday variation and matrix interferences were non-significant.. We acknowledge the helpful suggestions and technical support of Dr. James C. Smith, Jr., of the Veteran’s Administration Hospital, Washington, D.C., and we thank Texas Instruments, Inc., for their gift of the mini-computer used. REFERENCES 1 2 3 4 5 6 7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

24

F. W. Sunderman. Jr.. Human Pathol.. 4 (1973) 549. J. A. Haisted. J. C. Smith, Jr., and M. I. Irwin, J. Nutr., 104 (1974) 305. W. hlertz. Adv. Clin. Chem., 21 (1975) 468. A. Zettner. Clin. Chem.. 7 (1963) 1. A. Prasad, D. Oberleas, J. A. Haisted, and R. S. Collins, J. Lab. Ciin. Med., 66 (1965) 508. R. E. ‘Phiers, hlethods Biochem. Anal., 5 (1957) 273. S. Sprague and W. Shin. At. Absorpt. New&, 4 (1965) 228. B. hi. Hackley, J. C. Smith, Jr., and J. A. Halsted, Ciin. Chem., 14 (1968) 1. J. G. Reinhold, E. Pnscoe, and G. A. Kfoury, Anal. Biochem., 25 (1968) 557. B. hlomcilovic, B. Belonje, and B. G. Shah, Chin. Chem., 21 (1875) 588. S. Meret and R. I. Henkin, Ciin. Chem., 17 (1971) 369. J. B. Dawson and B. E. Waiker, Clin. Chim. Acta, 26 (1969) 465. R. S. Pekarek, W. R. Beisel, P. J. Bartelloni, and K. A. Bostonian, Am. J. Ciin. Pathol., 57 (1972) 506. J. D. Winefordner and H. W. Latz, Anal. Chem., 33 (1961) 1’727. N. A. Lange, (Ed.), Handbook of Chemistry, 9th edn. Handbook Publishers, Sandusky, Ohio, 1949. pp. 10-289. Clinical Methods for Atomic Absorption Spectroscopy, Perkin Elmer Corp., Norwalk, Conn., 1971, p. _&4-znl, 1. D. 0. Rodgerson and N. W. Tietz, Clin. Chem., 21 (1975) 1057. R. T. Lofbeq and E. A. Levri, Anal. Lett., 7(12) (1974) 775. B. Foley, S. A. Johnson, B. Hackley, J. C. Smith, Jr., and J. A Haisted, Proc. Sot. Exp. Biol. Med., 128 (1968) 265. G. D. Christian and F. Feldman, Atomic Absorption Spectroscopy, Wiley-Interscience. N. Y., 1970, p_ 206. A Goldstein, Biostatistics: An Introductory Text, Mnchlillan. N.Y., 1965, pp. 47. 59.81, 144. J. Harkness, Biorheology, 8 (1971) 171. J. F. Swindeils, R C. H&y, and R. L. Cottington. J. Res. Nat. Bur. Std., 52(3). C2479 (1954). N. Tietz, Fundamentals of Clinical Chemistry, W. B. Saunders, Philadelphia, 1970. p_ 934_