241
Clinica Chimica Acta, 96 (1979) 241-246 @ Elsevier/North-Holland Biomedical Press
CCA 1085
PLASMA SOMATOMEDIN CHILDREN
P.S. MOHAN
* and KAMALA
ACTIVITY
IN VITAMIN
A DEFICIENT
S. JAYA RAO
National Institute of Nutrition, Indian Council of Medical Research, Jamai-Osmania P.O., Hyderabad - 500 007 (India) (Received
February
16th,
1979)
Summary A role for vitamin A in the sulphation of mucopolysaccharides has been suggested. Somatomedin, a growth hormone dependent serum factor, has also been shown to stimulate the uptake of sulphate by cartilage. Therefore studies were undertaken on vitamin A deficient children to examine the possible interrelationship between vitamin A and somatomedin activity. Plasma somatomedin activity was markedly lowered in vitamin A deficient children and plasma HGH levels were in the normal range. The data suggest that vitamin A and somatomedin may be interrelated and also that plasma somatomedin activity may not always be determined by plasma growth hormone levels.
Introduction In young animals deprived of vitamin A, skeletal growth and remodelling process are retarded. An impairment in the sulphation and synthesis of mucopolysaccharides has also been demonstrated [ 11. It is now well established that sulphate uptake by cartilage is stimulated by a growth hormone dependent serum factor, somatomedin [2]. A study was therefore undertaken to estimate serum somatomedin activity in vitamin A deficient children. Subjects and methods Twelve children with ocular signs of vitamin A deficiency namely, conjunctival xerosis and Bitot’s spots were studied. Their ages ranged from 2 to 8 years. Nine apparently normal children drawn from similar socio-economic strata as the experimental group served as controls. Their ages ranged from 2 to 11 years. * To whom correspondence should be addressed
The weights and heig1~t.s of both thca study groups and the> controls wtbrfl lt>ss than the third percentile of the Harvard Standards [ 31. A sample of venous blood was obtained aft.er an overnight fast, under resting conditions. The children were then administered 24000 1.13. vitamin .;\ orally, daily for a period of 10 days. To avoid changes in thp intake of other nutrients, the children were not admitt,ed int,o the hospital, hut, were allowed to continue on their habitual home diets. At the end of 10 days, blood was again drawn under basal conditions. Blood was collected under heparin, plasma separated and stored at. --20°C. Care was taken to avoid exposure of the sample to light. Serum somatomedin activity was estimated by a four-point bioassay described by Yde [4]. However, each concentration of plasma was set up in triplicate. Both test and reference plasma were tested at 10% and 20% v/v. Pooled plasma from healthy human donors served as reference plasma. The final hydrolysis of the cartilage was carried out with papain as described by Alford et al. [ 51. Radioactivity in the hydrolysate was counted in a liyuid scintillation spectrometer, and expressed as cpm per mg dry weight of cartilage. The ratio of sulphate uptake obtained with test plasma to that obtained with reference plasma was calculated and this was referred to as sulphate uptake ratio. In six of the normal and six of the vitamin A deficient children the assay was also performed using four concentrations of plasma namely 5, lo,15 and 20% v/v. The ratio was calculated according to the classical method of Finney [6] and this is referred to as potency ratio. Dose-response lines obtained with samples from vitamin A deficient children were found to be parallel to those obtained on normal children and with reference plasma. Plasma growth hormone was estimated by the double antibody radioimmunoassay [ 71, The HGH and anti-HGH guinea-pig serum were kindly gifted by the NTAMDD of National Institute of Health, U.S.A. The first International reference preparation of HGH was used as the standard (1 mg = 2 I.U.). Precipitating antibodies were prepared by injecting rabbits with guinea-pig gamma globulins, Plasma vitamin A was estimated by the method of Neeld and Pearson [S] with slight modifications [9]. Plasma albumin was estimated by the biuret method [ 101. Results The mean plasma sulphate uptake ratio in normal children was 0.97 + 0.043 (Table I). The ability of serum to stimulate sulphate uptake by cartilage in vitro, expressed as sulphate uptake ratio, was found to be impaired in vitamin A deficiency (Table II). The value increased significantly following vitamin A therapy (P < 0.01). Neither age nor body weight appeared to influence the values (Tables I and II). The values expressed as potency ratio were also significantly low in the vitamin A deficient children (Table III). Both in normal and in vitamin A deficient children, values expressed either as sulphate uptake ratio or as potency ratio were essentially similar. The mean plasma vitamin A level in vitamin A deficient children showed a significant increase following treatment (P < 0.001). The mean plasma albumin
243
TABLE I PLASMA
SOMATOMEDIN
ACTIVITY
IN NORMAL
CHILDREN Plasma sulphate uptake ratio
Age
Height
Body weight
(years)
(cm)
(kg)
10 5 8 4 ‘/a 4 6 l/a 2 11 5
120 116 95 100 112 74 127 120
15.6 12.2 19.6 13.2 12.4 17.4 8.4 22.0 18.8
0.98 1.07 1.05 0.99 0.70 1.05 1.12 0.87 0.93
108.0 6.15
15.5 1.44
0.97 0.043
Mean S.E.M.
value was also low (P < 0.01) and there was no significant change after vitamin A therapy (Table IV). The mean plasma growth hormone level was not significantly different from that observed in normal children and no alterations were noted after vitamin A treatment (Table IV). No correlation was observed between plasma vitamin A and GH levels, or between plasma GH levels and sulphate uptake ratio or between plasma vitamin A levels and sulphate uptake ratio.
TABLE 11 PLASMA SOMATOMEDIN NO.
1 2 3 4 5 6 7 8 9 10 11 12
Sex
M F M F F F F F M F M M MIXIn S.E.M.
ACTIVITY
IN VITAMIN
A DEFICIENT
Age
Height
Body weight *
(years)
(cm)
(kg)
6 8 2% 5 2 5% 3 5 4% 6 3 5
106 68 80 68 98 79 98 87 101 92 91 88.0 3.89
CHILDREN Plasma sulphate uptake ratio Before treatment
After treatment
11.8 15.0 6.0 9.8 6.4 12.2 9.0 10.8 9.0 11.0 11.8 11.8
0.67 0.76 0.58 0.87 0.88 0.59 0.55 0.75 0.74 0.68 0.44 0.99
1.03 0.92 0.56 1.10 1.12 1.12 1.15 0.86 0.86 1.21 1.13 0.90
10.4 0.73
0.71 0.045
1.00 0.053
* Note: No changes in body weight were observed after treatment with vitamin A.
244
TABLE
III
COMPARISON TWO
BETWEEN
METHODS
PLASMA
Plasma
somatomrdin
(potency
(a) Normal
SOMATOMEDIN
ACTIVITY
children
activity
Plasma
1.07
0.87
0.88
1.47
1.57
1.05
0.97 1.04
1.09
1.15
S.E.
0.126
0.107
A deficient
children
sulphate
uptake
ratio
(6)
0.79
0.81
0.67
0.68
0.62
0.59
0.66
0.47
0.95
0.87
0.60
0.75
Mean
0.72
0.70
S.E.
0.054
0.060
TABLE
THE
1.36
0.91
0.78
* Methods
BY
(6)
Mean
Vitamin
CALCULATED
ratio)
1.48
(b)
VALUES
*
of calculation
described
in the
text.
IV Plasma
albumin
Plasma
(P”/) Vitamin
A deficient
Before After Normal
Values cance: 0.01.d
treatment (9)
are
mean
A
Plasma
GH
(ng/mI)
%)
Plasma
sulphate
uptake _-
ratio
(12)
treatment
before
(w
vitamin
+ S.E.M.
treatment
2.8
i 0.21
17.2
f 3.18
8.4
? 1.89
0.71
t 0.045
2.8
i 0.19
46.0
* 6.58
b
7.7
f
1.59
1.00
c 0.053
a
3.7
t 0.16
30.7
+ 3.19
c
8.8
* 3.03
0.97
* 0.043
d
Figures
vs. after
c
in parentheses
treatment:
a
indicate
number
P < 0.01,b P <
0.001:
of
children
normal
studied.
vs. vitamin
Statistical A deficient:
signific p <
P < 0.01.
Discussion The results of the present investigation show that the sulphate-uptake stimulating activity in plasma is lowered in vitamin A deficiency. An important substance in plasma which possesses this activity is somatomedin. The results show that values obtained by a four-point bioassay (expressed as sulphate uptake ratio) are essentially similar to those obtained by a classical bioassay as described by Finney [6]. This indicates that plasma sulphate uptake ratio as measured in the present study may reflect plasma somatomedin activity. In all the vitamin A deficient children studied here an increase in plasma sulphate uptake ratio was seen following treatment. The children were treated
245
only with vitamin A and to avoid changes in the intake of nutrients they were allowed to continue on their home diets. This was considered necessary because the children belonged to low socio-economic groups in which protein-energy deficiency is frequently seen and plasma somatomedin activity has been shown to be depressed in protein-energy malnutrition, too [11,12]. The increase in the ratio following treatment may therefore be considered to be due to vitamin A alone. It is not known how vitamin A deficiency may depress plasma somatomedin activity. A lowered activity has been reported in situations where plasma HGH levels are low [ 131. However in the present study the hormone levels were found to be in the normal range. Though somatomedins are growth hormone dependent factors, alterations in plasma somatomedin activity may not always follow the direction in which HGH levels change. For instance, plasma somatomedin activity is reported to be low during fasting [14] and in children with kwashiorkor [ 11,121, conditions where plasma HGH levels are either normal or high. Thus, factors other than growth hormone may also play a role in the regulation of somatomedin activity. Vitamin A is probably one such factor. One of the main sites of somatomedin generation is the liver [15]. It is not known whether the vitamin helps in the stimulation of somatomedin generation by growth hormone. Our preliminary studies on experimental animals indicate that this may not be so. It is possible that vitamin A per se may stimulate sulphate uptake by cartilage. Since somatomedin activity is measured by a bioassay, the observed activity will actually be the net result of all substances in plasma that stimulate or inhibit sulphate uptake. There are reports that vitamin A can exert its actions on tissues directly in vitro [ 16,171. It needs to be tested whether the lowered somatomedin activity is merely due to low plasma vitamin A levels or whether vitamin A stimulates somatomedin activity. Acknowledgements We wish to thank Dr. S.G. Srikantia, Director for his valuable advice and keen interest in this study. We also express our thanks to Drs. Vinodini Reddy and P. Bhaskaram for providing us the subjects for study. References 1 Wolf, G. and Varandani. P.T. (1960) Biochim. Biophys. Acta 43.501-512 2 Ha& K. (1972) Acta Endocrinol. (Suppl.) 163.1-51 3 Nelson, W.E., Vaughan. V.C. and McKay, R.J. (1969) in Textbook of Pediatrics, PP. 42-47. W.D. Saunders, Philadelphia, London 4 Yde. H. (1968) Acta Endocrinol. 57.557-564 5 Alford, L.P., BeIIair, B.T.. Burger. H.G. and Lovett, N. (1972) J. Endocrinol. 54.365-366 6 Finney, D.J. (1964) in Statistical Method in Biological Assay, 2nd edn.. Griffin, London 7 Pennisi, F. (1968) J. Nucl. Biol. Med. 12,137-138 8 Neeld, J.B. and Pearson, W.N. (1963) J. Nutr. 79.454-462 9 RoeIs, O.A. and Trout. M. (1972) in Standard Methods of Clinical Chemistry (CooPer. G.R. and King Jr.. J.S., eds.). Vol. 7, pp. 215-230, Academic Press, New York and London 10 Wolfson, W.Q., Cohn. G., CaIvory. E. and Ichiba, F. (1948) Am. J. Clin. Pathol. 18.723-730 11 Grant, D.B., Hambley. J., Becker, D. and Pimstone. B.L. (1973) Arch. Dis. Child. 48.596-600
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