462
BIOCHEMICAL SOCIETY TRANSACTIONS
Collins, P. B., Johnson, A. H. & Moriarty, M. (1980) Ir. J . Med.
Sci. 149 (8), 301-306
Deegan, K. G., Coulthart, S. W., Wualman, S. J. & Schork, M. A.
(1977) Cancer Res. 37, 44754481
Hallgren, H. M. & Yunis, E. J. (1977) J. Immunol. 118 (6), 20042008
Hallgren, H. M., Kersey, J. H., Dubey, D. P. & Yunis, E. J. (1978)
Janeway, C. A. (1982) Immunol. Today 3 (lo), 261-265
Lipsky, P. E., Ginsburg, W. W., Finkelman, F. D. & Ziff, M.
(1978) J . Immunol. 120 (3), 902-910
Toge, T., Yanagawa, E., Nakanishi, K., Yamada, Y., Nimoto, M.
& Hattori, T. (1980) Gum 71, 784-789
Weksler, M. E. (1980) Proc. SOC.Exp. Bwl. Med. 165, 20&205
J . Immunol. Immunopathol. 10, 65-78
Ascorbic acid content of plasma and cellular components of blood
ATHENE M. B. KEATINGE,*
BERNADETTE M. HANNIGAN,*
ALAN H. JOHNSON,* PATRICK B. COLLINS,*
MICHAEL MORIARTY,t and
PARNELL KEELING1
*Department of Biochemistry, Royal College of Surgeons in
Ireland, St. Stephen’s Green, Dublin 2, TSt. Luke’s Hospital,
Dublin 6, SJervis Street Hospital, Dublin 1, Ireland
There has been little attention paid to the precise distribution of ascorbic acid in the purified cellular components of
blood. In view of recent reports (Anderson et al., 1980;
Anthony & Scorah, 1982) that ascorbic acid is required for
immune responsiveness, a more detailed assessment of its
cellular distribution is indicated. Preliminary studies in our
laboratory (Keatinge et al., 1983) indicated that mononuclear leucocytes contained a higher concentration of
ascorbic acid than polymorphonuclear leucocytes. The aims
of this study were to compare the ascorbic acid content of
plasma, platelets, erythrocytes, lymphocytes, monocytes
and polymorphonuclear leucocytes in two healthy populations and in a group of patients suffering from malignancy.
The subjects were (a) a group of healthy medical students
Abbreviation used : PBS, phosphate-buffered saline.
(n = 15) aged 18-24 years (mean 21); (b) a group of patients
(n = 12) from Jervis Street Hospital, Dublin, aged 48-81
years (mean 69), who had been admitted for minor surgical
procedures and who were otherwise healthy; and (c) a group
of cancer patients (n = 22) from St. Luke’s Hospital, Dublin,
aged 54-90 years (mean 70), presenting for the first time before any treatment. The malignancy in the cancer patients
was diagnosed as squamous cell carcinoma at a variety of
sites.
Fasting blood samples (20ml) were obtained by venipuncture and placed in heparinized tubes. The blood was
separated into a supernatant of plasma (containing the
platelets) and a pellet of erythrocytes, mononuclear leucocytes and polymorphonuclear leucocytes. The platelets were
separated as described by Newman et al. (1978). The pellet
was resuspended in 2vol. of PBS, pH 7.0, and the cells separated by isopycnic centrifugation by the method of Boyum
(1968). Polyrnorphonuclear leucocytes were removed and
contaminating erythrocytes were lysed by incubation at
37°C with 2Ovol. of 0.83% (w/v) NH,C12 in 0.01~-Tris
buffer, pH 7.4. The mononuclear leucocytes were separated
into their constituent lymphocytes and monocytes by centrifugation on a Percoll gradient as described by Ulmer & Flad
(1972).
All cell preparations were washed and suspended in PBS
15C
......
..... ..
.....
.... z
..
--
1
v)
8
2
0
loo
M
a
4
v
m
.-
U
4
.-U
4
2
<
.
...
.....
.
.....
.
b
50
4
b
4
PS
.,
PT
ER
LY
MO
PO
Fig. 1. Ascorbic acid levels of plasma and blood cell fractions
Abbreviations used : PS, plasma; PT, platelets; ER erythrocytes; LY, lymphocytes;
MO, monocytes; PO polymorphonuclear leucocytes. Symbols : Q, young controls;
0,
old controls;
cancer patients. The results are mean values with bars to
indicate S.E.M.
1984
463
606th MEETING, CORK
with the exception of platelets which were treated with
O.O~M-EDTA
and 0. I5M-NaCI in 0.05hf-Tris buffer, pH7.4.
A small aliquot of each of the cell preparations was removed
for enumeration. The cell suspensions, together with an aliquot of plasma, were then diluted with trichloroacetic acid
to a final concentration of 5% (w/v). The samples were
thoroughly mixed and left overnight at 4°C. After centrifugation the acidic supernatants were removed and their ascorbic acid contents were estimated colorimetrically using
the dinitrophenylhydrazine procedure of Denson & Bowers
(1961).
The results for the ascorbic acid content of plasma, platelets, erythrocytes, lymphocytes, monocytes and polymorphonuclear leucocytes for the three subject groups are
shown in Fig. I . There is significantly more plasma ascorbic
acid in the young controls as compared with the old controls
( P <0.05) and similarly in the young controls as compared
with the cancer patients (P<O.O5). However, there was no
statistically significant difference between the two older
groups.
Ascorbic acid levels in elderly cancer patients appear to
be no different from a relatively healthy elderly group.
Therefore it appears that any decreases in the ascorbic acid
content of the plasma and the various cellular components
of blood are age-related rather than due to malignancy.
In the three subject groups fractionation of the white
blood cells into lymphocytes, monocytes and polymorphonuclear leucocytes reveals varying amounts of ascorbic acid
(as pg/108 cells) in the ratio of 6 :12 :1 (young controls),
4 :7 :1 (old controls) and 3 :5 :1 (cancer patients). It is possible that ascorbic acid is required by lymphocytes and
monocytes for immune competence and further work may
reveal whether this different intercellular distribution,
between the three groups, parallels the decrease in cellmediated immunity observed in similar subject groups
(Hannigan et al., 1983).
Anderson, R., Oosthizen, B., Maritz, R., Theron, A. & Van
Rensberg, A. J. (1980) Am. J. Clin. Nutr. 33, 71-76
Anthony, H. & Scorah, C. J. (1982)Br. J. Cancer 46, 354-367
Boyum, A. (1968) S c a d . J . Clin. Inuest. 21 (Suppl. 97), 9-29
Denson, K. W. & Bowers, E. F. (1961) Clin. Sci. 21, 157-162
Hannigan, B., Johnson, A. & Collins, P. (1983)Irish J . Med. Sci.
152, 254
Keatinge, A. M. B., Collins, P. B., Hannigan, B. M. &Johnson, A.
H. (1983) Proc. Inr. Congr. Food Sci. Technol. 6th 3, 2627
Newman, K.D., Williams, L. T., Bishopric, R.J. & Lefkowitz, R.
J. (1978) J. Clin. Invest. 61,395402
Ulmer, A. J. & Flad, H. (1979)J . Immunol. Meth. 30, 1-10
Effect of varied membrane protein density on the lateral diffusion of lipids in the
mitochondria1 inner membrane
BRAD CHAZOTTE, EN-SHINN WU* and
CHARLES R. HACKENBROCK
Laboratories j o r Cell Biology. Department of Anatomy,
School of Medicine, University of North Carolina,
Chapel Hill, NC 27514, U . S . A .
ment procedure permits the controlled incorporation of
various amounts of lipid into the inner membrane to give
membrane populations of different lipid/protein ratios.
To facilitate F R A P measurements, which require a
minimum 5 pm-diam. membrane, we have developed a
calcium fusion method to fuse the relatively small phosThe role of lateral diffusion of membrane components in the pholipid-enriched inner membranes into large spherical
mechanism of electron transport in the mitochondrial inner membranes up to 200pm in diameter. The fusion of the
membrane remains a fundamental question in mitochon- membranes was realized in the presence of a millimolar/
drial bioenergetics. On the basis of our previous studies we CaCI, solution buffered to a low pH (e.g. 6.5) at 37°C for
have demonstrated that the distribution of the electron- 15min on a specially constructed microscope slide. Kinetic
transport-chain components is random within the plane of measurements of enzyme activities at this temperature and
the membrane and have postulated that the factors affect- time duration did not show any significant deleterious
ing their rates of lateral diffusion must affect their inter- effects.
action (see, e.g., Hackenbrock, 1981).
Freeze-fracture electron microscopy of the calcium-fused
The concentration of the reactive components and their enriched inner-membrane-matrix revealed that intramemdiffusion coefficients are the two major parameters in any brane particles (integral proteins) were present in all memcollision-based mechanism. Therefore any variation in the branes observed and that their distribution was random in
protein density of the inner membrane should affect the rate the membrane plane. Furthermore, the intramembrane parof electron transport. We report here, as part of a systematic ticle density after calcium fusion was the same as that in the
study of the rates of diffusion of mitochondrial inner-mem- enriched membranes before fusion.
brane components and their respective kinetics, the lateral
The results of the FRAP measurements are presented in
diffusion of fluorescent lipid analogue d i I t in membranes of Table 1 as a function of phospholipid enrichment (lipid/provaried protein density by the technique of FRAP.
tein ratio). As a control a value of D = 3.3 x 10-9cmZ/sfor
Two types of mitochondrial inner membranes were used diI diffusion in mouse megamitochondrial inner membrane
in these studies. ( I ) Megamitochondria (used as a control) was determined in the absence of calcium fusion. This value
were obtained from the livers of cuprizone-fed mice and shows that low C a 2 + concentration (<lOmM) in the calwere hypo-osmotically swollen to a size large enough for cium-fused asolectin-enriched membranes has little or no
FRAP studies (Gupte et al., 1983). ( 2 ) Mitochondria1 inner- effect on dil diffusion. In asolectin-enriched membranes the
membrane-matrix particles (mitoplasts) were prepared diI diffusion increased significantly as the lipid/protein
from rat liver. Exogenous phospholipids (asolectin) were in- ratio increased. The same trend was seen when the memcorporated into the rat liver inner-membrane-matrix by the brane lipid composition was substantially altered by the use
low-pH method of Schneider et al. (1980). This lipid enrich- of an asolectin/cardiolipid mixture (9 : I , w/w) for enrich* On leave: Department of Physics, University of Maryland ment. The use of an asolectin/cholestero1 mixture (10 : 1.7
w/w) (cholesterol is not normally present in the mitochonBaltimore County. Baltimore, MD 21228, U.S.A.
t Abbreviations: dil, 3,3-dihexyldecylindoarbocyanine; drial inner membrane) showed a similar trend except at the
FRAP, fluorescence recovery after photobleaching.
highest cholesterol concentration, which showed a signifiVol. 12
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