The concept of carbohydrate as a protein-sparer is long established and

Transcription

1 J. Physiol. (1963), 165, pp With 6 textftgures Printed in Great Britain THE INFLUENCE OF DIETARY CARBOHYDRATE INTAKE ON SERUM PROTEIN LEVELS BY BETTY L. COLES AND I. MACDONALD From the Departments of Physiology, Royal Free Hospital School of Medicine, London, W.C. 1, and Guy's Hospital Medical School, London, S.E. 1 (Received 18 June 1962) The concept of carbohydrate as a proteinsparer is long established and it would be expected that the addition of carbohydrate to lowprotein diets would result in an amelioration of the effects of protein malnutrition. The experiments to be described were designed to test the effect on the serum proteins of varying the carbohydrate content of a diet of fixed lowprotein intake. The type of carbohydrate used was also varied. The results suggest that under certain circumstances carbohydrates seem to have an effect which is the reverse of proteinsparing. METHODS Twentytwo adult rabbits of approximately 2 kg weight were given a diet consisting mainly of stock green food in powder form, to which was added varying amounts of different carbohydrates. The carbohydrates used were maize starch, dried liquid glucose B.P.C., and sucrose. The composition of the diet and method of feeding were so arranged that paired animals of similar weight ate the same small amount of protein but varying amounts of carbohydrate (Macdonald, 1962). The carbohydrate was fed at two levels, 'high', 65% by weight of the dry diet and 'low' 36 % of the dry diet. The animals were killed when a third of the initial body weight had been lost. Serum samples were examined at threeweekly intervals, blood being obtained from the marginal vein of the ear. The serum proteins were separated by paper electrophoresis in a vertical tank with Whatman No. 31 extra thick paper. Samples of 0 05 ml. serum were run for 16 hr at a current of 025 ma/cm. width of paper. A barbitone:sodium barbitone buffer of ph 86 was used throughout. The strips were dried at 1100 C and stained with 001 % bromophenol blue after the method of Block, Durrum & Zweig (1955). The indidivual protein fractions were then estimated by the method of Levin & Oberholzer (1953), and the sum of the fractions was checked against the total serum protein as estimated on 01 ml. samples with a microkjeldahl technique. All results were expressed as g protein/100 ml. serum. RESULTS The results have been expressed as the slope of the regression line of length of time on the diet (in weeks) plotted against the concentration of the protein in g/100 ml. serum. Findings are considered statistically significant when P = < Physiol. 165

2 328 BETTY L. COLES AND I. MACDONALD Total protein fell in all groups except the animals on liquid glucose with low carbohydrate intake. The rate of fall was greater in the animals fed on lowstarch diets than in those on the highstarch intake, but with liquid glucose and sucrose the reverse occurred. A greater rate of fall was found in groups fed on high sucrose or liquid glucose than in those fed on highstarch diets. With the low level of carbohydrate feeding the rate of fall was greater in animals fed on starch than in those fed on sucrose or liquid glucose. 4 Starch ~~~~(P <001) 2 ~~~(P= <0001) _ 1 _ 4 Liquid glucose E 8 3 b0 _r (P= <0001) E o E1 Sucrose 2_ (W= <0001) I I I L Fig. 1. The mean response of serum albumin level to high and low intakes of starch, liquid glucose B.P.C., and sucrose. In Figs. 14, low carbohydrate diet, high carbohydrate diet. Albumin. The results for albumin are shown in Fig. 1. This fraction fell significantly in every group except the lowlevel sucrose and liquid glucose diets. acglobulin8. The only significant fall was in the animals on liquid glucose at high intakes (Fig. 2).

3 DIETARY CARBOHYDRATE AND SERUM PROTEIN 329 fglobulins. Falls in the concentration of this fraction occurred in animals on high intakes of liquid glucose and low intakes of starch and sucrose (Fig. 3). yglobulins. No significant change in the concentration of this fraction was seen in any group (Fig. 4). The results, which are summarized in Table 1, show that significant changes were mainly confined to the serumalbumin fraction and the changes seen in the total protein were due to a fall in this fraction. If all 06Starch 06 : Starch S= 0 4 _ 024 E 0.9 Liquid glucose E 0 6 = Liquid glucose a ~ j C~~~~~~~~~~~~~~~C.0~~~~~~~~P= S.0 E 0.9 _Sucrose E Sucrose <, 04 02~~~~~~~~~~~~~~. 0.2 _ Ns ffi ~~~ ~~0.31 l Fig. 2 Fig. 3 Fig. 2. The mean response of serum aglobulin level to high and low intakes of starch, liquid glucose B.P.C., and sucrose. Fig. 3. The mean response of serum sglobulin level to high and low intakes of starch, liquid glucose B.P.C., and sucrose. the serumalbumin levels for every type of carbohydrate diet at each 3 weekly interval were plotted against the mean daily carbohydrate content of the diet (expressed as g/kg initial body wt./24 hr), it was found that up to a critical level the higher the carbohydrate intake the lower the serumalbumin level. At a daily intake of about 14 g carbohydrate/kg initial body wt./24hr this relationship seemed to change; the serumalbumin levels improved with increase in carbohydrate intake, but never reached the original values. Figure 5 shows these findings graphically for the 3rd, 212

4 0 6 _ ~~~~~~~Starch 330 BETTY L. COLES AND I. MACDONALD 6th, 9th and 12th weeks of the experiment. It would appear that on these restricted protein diets the inclusion of carbohydrate in the diet exerted a depressing influence on the serumalbumin level, reaching its maximal effect about the level 14 g/kg, and beyond that additional carbohydrate seemed to be progressively less depressing, i.e. the values of serum albumin began to rise. Figure 6 attempts to illustrate the influence of time upon these effects by plotting the slopes of lines in Fig. 5 against their respective experimental timing. The smoothness of this graph suggests some steadily progressive influence of dietary carbohydrate upon the serumalbumin formation; up to 14 g/kg initial body wt./24 hr the influence was entirely depressing, above 14 g the influence was reversed o ~ 0 so 0.4 E 08 Liquid glucose Sucrose 06 Fig The mean response of serum yglobulin level to high and low intakes of starch, liquid glucose B.P.C., and sucrose. TABLE 1. Changes in serum protein on experimental diets Liquid Starch glucose Sucrose Carbohydrate A, I Intake level... High Low High Low High Low Total protein Albumin ocglobulin ,Globulin yglobulin Significant fall in protein level during time on diet; 0 = no change in protein level.

5 DIETARY CARBOHYDRATE AND SERUM PROTEIN (P= <0.05) * * SeS~~~ 0 * 3/52 o E 4*0 32 _ M (P=0025) a* 0 so6/52 av 32 _ 24 4*0 32 _ 24 Normal ±D * + S.D. 0 * (P0*025) *. 0 0.E_ 40 0 In 0I * II 152 0~~~~ 12/52 l l j Carbohydrate (g/kg initial body wt./24 hr) Fig. 5. The serumalbuimin level plotted against carbohydrate intake (as g carbohydrate/kg initial body wt./24 hr) at varying periods after the commence. ment of the experiment. +008r (P= <0025) j.c.l.s v0 04 = v D e.0 V ou, 012 Gareater '\. NN14 g. carbohydrate/.i kg initial body wt./24 hr NN ON, Less N ""N N\ (P== <0.005)N I _ {Jlh ' ' ' Fig. 6. The slope of the line serumalbumin level versus carbohydrate intake plotted against length of time of the diet, for carbohydrate intakes above and below 14 g/kg initial body wt./24 hr. 12

6 BETTY L. COLES AND I. MACDONALD DISCUSSION The daily protein intake necessary for maintenance of health in a 2 kg rabbit is 4 g/kg (Smith, Templeton & Weir, 1954), and it can be seen in Table 2 that the rabbits used in this experiment had a protein intake considerably below this level. The animals fed on starch at both high and low levels ate more per kilogram than those fed on the other carbohydrates. TABLE 2. Mean daily intake of protein and carbohydrate (g/kg initial body wt.) Carbohydrate Starch Liquid glucose Sucrose Intake level... High Low High Low High Low Protein intake Carbohydrate intake F This led to a larger, yet still deficient protein intake by the starchfed group, which may account for the fact that the serum albumin fell less rapidly on a highstarch intake than on a highliquid glucose or sucrose diet. With low carbohydrate intake, however, the situation was reversed and the fall was more rapid in the starchfed animals. A possible explanation for this can be found in the behaviour of the serum albumin at various levels of carbohydrate intake. The results showed that on daily intakes of up to 14 g carbohydrate/kg initial body wt., increasing the amount of carbohydrate increased the fall in serum albumin regardless of the type of carbohydrate fed, but that at levels of intake exceeding 14 g the fall was less. This would suggest that the proteinsparing action of carbohydrate can only be detected above certain levels of carbohydrate intake. The greater fall in serum albumin with increasing daily carbohydrate intake up to 14 g/kg initial body wt. (Fig. 5) suggests that the dietary carbohydrate under these circumstances is having an effect which is the reverse ofproteinsparing. It may be that on these lowprotein diets the animal is forced to use a good deal ofproteinto satisfyits energy needs, and serum protein falls in consequence. Up to intake levels of carbohydrate of 14 g/kg initial body wt./ 24 hr, carbohydrate appears to exert a harmful effect and the fall in serum albumin is accentuated. Above 14 g/kg, carbohydrate begins to contribute to proteinsparing with the result that the fall in serum protein is progressively less marked. This credits the carbohydrate with a dual role in relationship to protein metabolism: at low levels of carbohydrate intake, its influence is to exaggerate protein deficiency, but above a certain critical level it begins to demonstrate the qualities of a proteinsparer. This critical level of 14 g/kg initial body wt./day may be related to the metabolic rate of the animal. It is known that when the energy intake falls below the normal output values, the output value falls. It is possible,

7 DIETARY CARBOHYDRATE AND SERUM PROTEIN 333 therefore, that increasing an energy intake that is below normal requirements, as by adding more carbohydrate, will increase the energy output towards the normal value and this will make greater demands on the dietary protein, which in these experiments remains fairly constant. Support for this view comes from the knowledge that the basal metabolic rate in normal adult rabbits is 46 kcal/kg/day (Albritton, 1954), a value equivalent to 1112 g carbohydrate, whereas in these experiments 14 g carbohydrate seems to be the critical value, and intakes up to 14 g/kg initial body wt./day worsen the serumalbumin level. A possible explanation for this may be that the increase in metabolic rate associated with an increase in lowcalorie intakes leads to greater utilization of body proteins and the dietary protein intake is not increased at a corresponding rate. At intakes above 14 g carbohydrate/kg the basal metabolic needs are more than met by the carbohydrate and no further increase in metabolic rate occurs; thus no further demands on the protein are made. Munro, Black & Thomson (1959) have suggested that carbohydrate causes deposition of amino acids in the muscles, with reduction of liver protein. The liver is generally accepted as the site of serumalbumin production and the above suggestion might explain the increased rate of fall of serum albumin in animals fed on the higher carbohydrate diets at suboptimal intakes. At daily carbohydrate intakes of 14 g/kg body wt. or over it is possible that the proteinsparing action is sufficient to overcome the local effects of diversion of protein from the liver to muscle, and the net fall of the albumin is checked owing to the increase of total available nitrogen in the body. Heard, Platt & Stewart (1958) found that pigs fed on a 5 % protein diet with a carbohydrate supplement of a starchglucose mixture showed a greater fall in total serum proteins and albumin than pigs fed on a 5 % protein diet without the extra carbohydrate. They gave no data for the other serum protein fractions. In underdeveloped countries where protein deficiency is very common, especially in infancy, protein malnutrition is frequently accompanied by a relative excess of carbohydrate, although the total calorie intake may remain well below normal. The resulting clinical condition has many names according to the locality, but is perhaps best known as kwashiorkor. This condition is associated with marked falls in the serumalbumin levels, whereas in the condition of marasmus, due to protein deficiency in association with a balanced diet, the changes in serum albumin are much less marked. In this series of experiments definite changes in the serum globulins were not found. It has been suggested (Brock & Autret, 1952) that the increases in a and fglobulins seen in children with kwashiorkor may be due to coincidental infections or infestations, both common complicating

8 "BETTY L. COLES AND I. MACDONALD factors in areas where the condition is prevalent. Decreases in serum albumin, however, are frequently accompanied by a rise in acglobulins, whatever the primary cause of the lowered albumin (Chow, 1947); but in these rabbits the tendency was for the ocglobulin to fall, though this was only significant in one group of animals. Proteindepleted animals in which there is no dietary imbalance of protein and carbohydrate also show an increase in oglobulin associated with a lowered albumin. Zeldis, Alling, McCoord & Kulka (1945) have shown this to be associated with increased plasma lipid levels. In the dog the greater amount of plasma lipid is associated with the oaglobulins rather than with the,8globulins as in man. Similar changes in the serum proteins have been reported in hypoproteinaemic pigs by Cartwright, Smith, Brown & Wintrobe (1948), again with hypoproteinaemia unaccompanied by any relative dietary carbohydrate excess. In rabbits the acglobulin separates electrophoretically as only one fraction, whereas in dogs, pigs and many other species two or more fractions can be distinguished. In addition, the lipoprotein content of the serum is low and its electrophoretic separation poor. One lipoprotein peak in the rabbit is seen at the origin, the second, ill defined and sometimes absent, occurs in the region of the acglobulin (Morris & Courtice, 1955). As increased aglobulin levels are associated with increased plasma lipids it appears likely that the failure of the rabbit to produce an increase in acglobulins under these experimental conditions is connected with this low lipid concentration and that this difference in response, compared with that seen in man, dogs and pigs, is a true species difference. SUMMARY 1. Experiments on the effect of addition of carbohydrate to lowprotein diets show that such additions generally exaggerate the fail in serum albumin. An analysis of the results suggests that carbohydrate can exert two opposing influences. 2. At lower levels of carbohydrate intake, an increase in the carbohydrate consumed resulted in a greater fail of serum albumin, an effect which would seem to be the reverse of proteinsparing. 3. Proteinsparing by carbohydrate as judged by the serum albumin level, was apparent only when the calories from the dietary carbohydrate reached a critical level which probably satisfied the animals energy requirement. 4. Other serum protein fractions showed no consistent changes. This work was done with a grant from the Endowments Fund, Guy's Hospital, London, S.E. 1. We are grateful to Dr R. J. L. Allen of Beechams Foods Ltd. for providing the rollerdried liquid glucose.

9 DIETARY CARBOHYDRATE AND SERUM PROTEIN 335 REFERENCES ALBRITTON, E. C. (1954). Standard Values in Nutrition and Metabolism, p Philadelphia: W. B. Saunders. BLOCK, R. J., DURRUM, E. L. & ZWEIG, G. (1955). A Manual of Paper Chromatography and Paper Electrophoresis, p New York: Academic Press. BROCK, J. F. & AUTRET, M. (1952). Kwashiorkor in Africa. Geneva: W. H. 0. Monograph Series No. 8. CARTWRIGHT, G. E., SMITH, E. L., BROWN, D. M. & WINTROBE, M. M. (1948). Electrophoretic analyses of sera of normal and hypoproteinaemic swine. J. biol. Chem. 176, CHOW, B. F. (1947). The correlation between the albumin and alpha globulin content of the plasma. J. clin. invest. 26, HEARD, C. R. C., PLATT, B. S. & STEWART, R. J. C. (1958). The effects on pigs of a low protein diet with and without additional carbohydrate. Proc. nutr. Soc. 17, xli. LEVIN, B. & OBERHOLZER, V. G. (1953). Paper electrophoresis of serum proteins with micro Kjeldahl nitrogen analysis of protein fractions. Amer. J. clin. Path. 23, MACDONALD, I. (1962). Some influences of dietary carbohydrate on liver and depot lipids. J. Phy8iol. 162, MORRIS, B. & COURTICE, F. C. (1955). The protein and lipid composition of the plasma of different animal species determined by zone electrophoresis and chemical analysis. Quart. J. exp. Physiol. 40, MiNRo, H. N., BLACK, J. C. & THOMSON, W. S. T. (1959). The mode of action of dietary carbohydrate on protein metabolism. Brit. J. Nutr. 13, SMITH, S. E., TEMFLETON, G. S. & WER, W. C. (1954). Recommended nutrient allowances for domestic animals. 9. Rabbits 112. U.S. Agricultural Board. ZELDIs, L. J., ALLING, E. L., MCCOORD, A. B. & KULKA, J. P. (1954). Plasma protein metabolism. Electrophoretic studies. Chronic depletion of circulation proteins during low protein feeding. J. exp. Med. 82,