840

METHOD

I

Determination of/?-Carotene in Plasma, Blood Cells and Buccal Mucosa by Electrochemical Detection Takuji Murata a, Hiroshi Tamai a,*, Takao Morinobu a, Mitsuhiro Manago a, Akira Takenaka b, Hiroyuki Takenaka c and Makoto Mino a aDepartment of Pediatrics, Osaka Medical College, Takatsuki, Osaka, bDepartment of Obstetrics and Gynecology, Shiga University of Medical Science, Ohtsu, Shiga and CNikken Sohonsha Co. Ltd., Gifu, Japan

The B ~ o t e n e concentrations in plasma, blood cells and buecal mucosal ceils were determined by high-performance liquid chromatography with electrochemical detection. This method was 1,000 times more sensitive than the conventional spectrophotometric method. Polymorphonuclear cells and red blood cells had lower B-carotene levels than the other cells. After oral administration of 580 mg/day of all-trans B~arotene to human male volunteers for a week, the B~arotene concentrations in all cell types increased at least several times above the original levels. Lipids 27, 840-843 (1992). The risk of developing cancer has been reported to be inversely correlated with the dietary intake of f~,~amtene (1-3), which is the major precursor of vitamin A among car~ tenoids. ~Carotene in part converts to vitamin A in the intestinal mucosa (4,5), while most of it is absorbed and re~ mains in the cireulatiorL There have been some reports (6-8) on the transport of B~arotene after ingestion in humans. Although B~carotene is known to be as efficient a singlet oxygen quencher as is lycopene (9,10), to our knowledge there have been few reports (11-13} on the physiological effects of/~-carotene in association with antioxidants. Alsa few reports have appeared on the B~arotene levels in body cells (14-16), which are quite low for being measured by conventional methods. Several recent studies have demonstrated the advantages of high-performance liquid chromatography (HPLC), coupled with electrochemical detection (ECD), for the determination of tocopherols (17,18), asco~ bic acid (19) and thiols (20). In the present study, we were able to determine very low levels of B~arotene in biological samples by using HPLC coupled with ECD. Furthermor~ changes in B-carotene levels were investigated after administration of a high-dose of oral B-carotene to adult humans.

MATERIALS AND METHODS

Sample preparation. Three healthy male volunteers (age 25 to 38) were enrolled in this study and given 580 mg of all-trans B-carotene]day for one week. All-trans B-carotene was a gift from Nippon Roche Ltd. (Toky~ Japan). None of the volunteers took any other vitamin supplements or medication during the study. The B-carotene levels in red blood cells (RBC), plasma~ platelets, mononuclear cells (MN), polymorphonuclear cells (PMN) and buccal mucosal cells (BMC) were analyzed before and at the end of ~-carotene administration. Heparinized blood was collected after an overnight fast. RBC, plasma and

platelets were obtained by the method reported previously (18). MN and PMN were separated by densitygradient centrifugation using "monopoly resolving medium | (ICN Biomedicals, Asse-Relegem, Belgium} (21). The cells were then washed three times with normal saline before being used for the B-carotene analysis. BMC were collected by gently scraping off the buccal mucosa with a spatula (18). The scraped-off cells were suspended in normal saline washed three times with normal saline and resuspended in distilled water. The suspension was vortexed and then sonicated at 20 KHz for 30 s. Aliquots of the suspension were taken separately for the B-carotene and protein assays (22). The protocol of the study was approved by the Ethics Committee of the hospital and was performed after informed consent was obtained from each of the subjects. Analysis of ~-carotene. One mL of ethanol containing 0.15% butylated hydroxytoluene (BHT) was added to 0.4 mL of plasma or cell suspension, followed by vigorous shaking in a nitrogen atmosphere Five mL of n-hexane was added to this mixture, which was then centrifuged at 3,000 rpm for 10 min; then 4 mL of the hexane layer was evaporated under a stream of nitrogen. The residue was dissolved in 50 ~L of ethanol, and a 20-~L aliquot was injected into the HPLC column. For the separation, an Irika Z 871 HPLC instrument (Irika Co. Ltd., Kyot~ Japan} with a Vydac reverse phase C18 column (250 X 4.6 mm; Vydac, Hesperia, CA) was used; the detector was an Irika 5 875 amperometric detector. The standard eluent was methanol/acetonitrile {95:5, vol/vol), which included 50 mM NaC104, and the flow rate was 1 mL/min. Authentic B-carotene was obtained from Nippon Roche K.K. (Toky~ Japan) and dissolved in ethanol for use as standard. The purity of the standard was checked by HPLC; at least 98% of the detectable carotenoid was eluted as a single fraction. The concentration of the authentic Bcarotene standard was determined with a Hitachi U-2000 spectrophotometer (Hitachi Co. Ltd., Tokyo, Japan) using a molecular coefficient Ecru % -- 2620 at 453 nm in ethanol.

RESULTS AND DISCUSSION

Method of~-carotene determination. ECD is possible on-

ly for substances supporting an electrolytic reaction within an applied voltage range. A typical chromatogram of an extract of plasma obtained from a volunteer without ~-carotene supplementation is shown in Figure 1 (top). The eluent was methanol/acetonitrile (95:5, vol/vol) with 50 mM NaC104. The arrow shows the putative ~-carotene *To w h o m correspondence should be addressed at Department of peak with a retention time identical to that of an authentic B-carotene standard. The voltamogram of plasma ~Pediatrics,2-7 Daigakumachi, Takatsuki, Osaka, Japan. carotene was consistent with that of the standard BAbbreviations:BHT, butylatedhydroxytoluene;BMC, buccalmucosal cell; ECD, electrochemicaldetection;HPLC, high-performanceliquid carotene as shown in Figure 2; both were oxidized at 0.6 chromatography; MN, mononuclearcell; PMN, polymorphonuclear V. When the eluent was changed, the presumed B-carotene cell; RBC, red blood cell. peak moved in accordance with the mobility of the stanLIPIDS, Vol. 27, no. 11 (1992)

841

METHOD

Sample

A

Sample

Standard

6O

f ==*0

~

L

(3

ne

20

0

B

Sample

Standard o

J

v

/

i,o

Standard

/

I~-carotene

c

Sample

Standard

,f 6O

FIG. 1. ~ p i c a l chromatograms of plasma and standard fl-earotene. The arrows point at the fl-carotenepeaks. The E-carotene peaks were symmetrical and well separated from other peaks.

/ 7~

140

2O

Standard O~O 12 0

(n

~

100

Retention T i m e

t::: (13 so J::: t~ 60 ,.~

(rain)

40

~: ~~ ~

,

,

400

,

,

,

500

,

600

,

,

700

80O

mV

FIG. 2. Voltamograms of sample and standard fl-carotene. The voltamogram curve of standard fl-carotenewas consistent with that of plasma; both were oxidized at 0.6 V. The X-axis shows the relative changes in current against those at 0.6 V.

dard fl-carotene (Fig. 3). Based on this, the putative flcarotene peak was established as being due to fl-carotene The concentration and peak area correlated in linear manner throughout the fl-carotene concentration range from 0 to 100 ng. The detection limit for/~carotene (a peak area greater than 10 times the background) was 15 pg, making this assay 1,000 times more sensitive t h a n previous methods based on spectrophotometric detection (Fig. 4}. The sensitivity was sufficient to determine flcarotene levels in various biological samples. Five replicate samples from pooled plasma were analyzed. The mean concentration was 0.195 ~g/mL, and the

FIG. 3. Effects of eluent composition on the mobility of/~-carotene in high-performance liquid chromatography. Panel A shows a chromatogram of plasma when the eluent was composed of methanol/acetonitrile (80:20, vol/vol) with 50 mM NaCIO 4. The presumed fl-carotene peak was not separated from other peaks. With an increase in the methanol concentration of the eluent (B; 90:10, C; 95:5), the surrounding peaks moved and the/3-carotene peak appeared as a single symmetrical peak. The peak also moved in accordance to the mobility of the standard fl-carotene.

intra-batch coefficient of variation was 3.6%, as shown in Table 1. When the recovery test was done for this assay, a satisfactory 95.3% recovery was measured as shown in Table 2. Determination of cellular f~varotene levels. We measured the concentrations of f~carotene in plasma, blood cells and BMC from volunteers which did not receive a supplement of fl-carotene Typical chromatograms are shown in Figures 1 and 5. Although the fl-carotene levels of these samples were very low, our assay was sensitive enough to detect them. When conventional detection was used for the red blood cell (14) and leukocyte (15) assays, f~carotene could be detected, but it could not be quantified. The/?-carotene concentrations in the cells and in plasma from the three male volunteers are shown in Table 3. After oral administration of 580 m g of fl-carotene/day for one LIPIDS, Vol. 27, no. 11 (1992)

842

METHOD

60

Standard Curve by Spectrophotometer Soo

Platelet

~

4o

100

20 0

100

2QO

300

/-carotene (ng)

Standard Curve by ECD

Retention Time (mln)

3O

FIG. 5. ~ypical chromatogram of a platelet sample. The arrow points

at the/3-carotenepeak./~arotenealsowas detectedin.redbloodcells, mononuclear cellsand buccal mucosa.

"E

TABLE 3 0

i f

l~Ca~

Changes in the fJ-Carotene Concentration in Various Biological Samples after Oral f~Carotene Administration a 2

(ng)

Before

FIG. 4. Calibration curves for st~mdard f~carotene by electrochemical detection (ECI)) and spectrophotometric detection. The limit of detec~ tion by the spectrophotometric method was 15 ng of/~carotene, and that for ECD was 15 pg. TABLE 1

Intra-Batch Coefficient Variance of ~-Carotene in Plasma Samples fl-Carotene in plasma pool samples (~g/mL) 1 0.190

2 0.187

Mean

0.195 ~g/mL 0.007 ~g/mL 3.612%

SD CV a

3 0.198

4 0.205

5 0.196

TABLE 2 Recovery of/3-Carotene in Plasma Pool Samples

/~-Carotene in plasma Added ~-carotene Observed f~-carotene Recoverya (~g/mL) (~g/mL) (~g/mL) (%) 0.052 0.055 0.108 0.112 0.158 0.161

0.230 0.230 0.280 0.278 0.348 0.348

95 95 93 89 101 99

aAverage 95.3.

week, the/~-carotene concentrations in plasma, platelet, B M C , M N , P M N and R B C increased to 35-, 6-, 8-, 4-, 3and 7-fold, respectively. The difference in incremental changes in ~carotene levels are likelyto depend on variations in lifespan, turnover and lipid content of the cells. Further studies are needed to determine the factors that LIPIDS, Vol. 27, no. 11 (1992)

0.19 5.05 0.79 0.71 0.20 1.51

+- 0.03 + 1.26 +_ 0.26 +_- 0.28 _ 0.09 • 0.74

After 6.85 37.30 6.23 2.68 0.58 10.2

+-- 0.16 + 7.70 • 0.84 • 0.66 • 0.11 • 8.5

~gtmL ng/mg protein ng/m~ protein ~g/10 ~ cells ~g/109 ng/mL packed cells

avalues are mean +__ SEM (n -- 3). Abbreviations: BMC, buccal mucosal cells; NM, mononuclear cell; PMN, polymorphonuclear cell; RBC, red blood cell.

influence the incremental changes in/3-carotene levels in biological samples. REFERENCES

aCV, coefficient variance.

0.190 0.188 0.193 0.202 0.186 0.192

Plasma Platelets BMC MN PMN RBC

1. Peto, R., Doll, R., Buckley, J.D., and Spore, M.B. (1981) Nature 290, 201-208. 2. Krinsky, N.I. (1988) Clin. Nutr. 7, 107-112. 3. Menkes, M.S., Comstoch, G.W., Vuilleumier, J.P., Helsing, K.J., Rider, A.A., and Brookmyer, R. (1986) J. Med. 315, 1250-1254. 4. Singh, H., and Cama, H.R. (1974) Biochim. Biophys. Acta 370, 49-61. 5. Villard, L., and Bates, C.J. (1986) Br. J. Nutr. 56, 115-122. 6. Dimitrov, N.V., Meyer, C., Ullrey, D.E., Chenoweth, W., Michelakis, A , Malone, W., Boone, C., and Fink, G. (1988) Am. J. Clin. Nutr. 4~ 298-304. 7. Brown, E.D., Micozzi, M.S., Craft, N.E., Bieri, J.G., Beecher, G., Edwards, B.K., Rose, A., Taylor, P.R., and Smith, Jr., J.C. (1989) Am. J. Clin. Nutr. 49, 1258-1265. 8. Gilbert, A.M., Stich, H.E, Rosin, M.P., and Davison, A.J. (1990) Int. J. Cancer. 45, 855-859. 9. Burton, G.W., and Ingold, K.U. (1984) Science 224, 569-573. 10. Mascio, P.D., Kaiser, S., and Sies, H. (1989) Arch. Biochem. Biophys. 274, 532-538. 11. Vile, G.E, and Winterbourn, C.C. (1988)FEBS Lett. 23~ 353-356. 12. Moore, M.M., Breedveld, M.W., and Author, A.P. (1989) Arch. Biochem. Biophys. 270, 419-431. 13. Krinsky, N.I. (1989) Free Radical Biol. and Med. 7, 617-635. 14. Mathews-Roth, M. (1975) Clin. Chem. 21, 258-259. 15. Mathews-Roth, M. (1978) Clin. Chem. 24, 700-701.

843

METHOD 16. Culling-Berglund, A.J., Newcomh, S~A.,Gagne, M., Morfitt, W.S~, and Davis, T.R (1989) J. Micronutr. Anal. 5, 139-148. 17. Deschuytere, A., and Deelstra, H. (1986) Fresenius Z. Anal Chem. 324, 1-4. 18. Tamai, H., Manago, M., Yokota, K., Kitagawa, M., and Mino, M. (1988) Int. J. Vit. Nutr. Res. 58~ 202-207. 19. Pachla, L.A., and Kissinger, PYr. (1979) Anal. Chem. 48, 364367. 20. Allison, L.A., Keddington, J., and Shoup, R.E. (1983) J. Liq. Chrornatogr. 6, 1785-1798.

21. Ferrante, A., and Thong, Y.H. (1978) J. ImmunoL Methods. 24, 389-393. 22. Lowry, O.H., Rosanbrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. BioL Chem. 193, 265-275.

[Received March 6, 1992, and in revised form July 24, 1992; Revision accepted August 9, 1992]

LIPIDS, Vol. 27, no. 11 (1992)

Determination of beta-carotene in plasma, blood cells and buccal mucosa by electrochemical detection.

The beta-carotene concentrations in plasma, blood cells and buccal mucosal cells were determined by high-performance liquid chromatography with electr...
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