The Journal of Nutrition Vol. 128 No. 2 February 1998, pp. 214-219

Repletion of the Plasma Pool of Nutrient Transport Proteins Occurs at Different Rates during the Nutritional Rehabilitation of Severely Malnourished Children^1,2,3

John F. Morlese^*, Terrence Forrester, Melanie Del Rosario^*, Margaret Frazer^*, and Farook Jahoor^{*, 4}

^*  USDA/Agricultural Research Service, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX. 77030 and  Tropical Metabolism Research Unit, University of the West Indies, Mona, Kingston 7, Jamaica

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Increased morbidity and mortality are associated with lower plasma protein concentrations in children with severe protein-energy malnutrition. However, the kinetic changes responsible for repletion of the plasma pools of nutrient transport proteins and the rapidity of their replenishment in these children have not been determined. This study was undertaken to determine whether an increased rate of synthesis is the mechanism responsible for repletion of the plasma retinol-binding protein, transthyretin and high density lipoprotein-apolipoprotein A1 concentrations of children with severe malnutrition during nutritional rehabilitation. The plasma concentrations and synthesis rates of retinol-binding protein, transthyretin and high density lipoprotein-apolipoprotein A1 were measured using a constant intragastric infusion of ²H₃-leucine in 22 children with severe protein-energy malnutrition, at ~2 d postadmission (study 1), ~8 d post-admission when infections were under control (study 2) and ~59 d postadmission at recovery (study 3). In study 1 the plasma concentrations and rates of synthesis of all the proteins were lower compared with values at recovery. In study 2, retinol-binding protein and transthyretin concentrations and absolute synthesis rates increased to the recovered values seen in study 3, but the high density lipoprotein-apolipoprotein A1 concentration and synthesis rate remained significantly lower. These results suggest that repletion of the plasma pool of these three nutrient transport proteins occurs at different rates, through an increase in the rate of synthesis.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Protein-energy malnutrition (PEM)⁵ is characterized by low plasma concentrations of the nutrient transport proteins transthyretin (TTR), retinol-binding protein (RBP) and high density lipoprotein-apolipoprotein A1 (HDL apoA1) (Ingenbleek et al. 1975, Smith et al. 1973, Toure et al. 1990). Transthyretin binds and transports thyroxine and also forms a protein-protein complex with RBP that binds and transports vitamin A as retinol from the liver to peripheral cells (Kanai et al. 1968, Putnam 1988, Raz et al. 1970). HDL apo A1, on the other hand, transports cholesterol from peripheral tissues to the liver (Barbaras et al. 1987). The increased morbidity and mortality associated with the lowered plasma protein concentrations of children with PEM (McLaren et al. 1969) underscore the importance of these proteins. In the malnourished person, a reduction in the plasma proteins responsible for the transport of nutrients to sites of utilization will further impede nutrient use; hence, the rate of recovery from PEM is closely related to replenishment of the plasma protein pools. To date, the kinetic changes responsible for repletion of the plasma pools of most plasma proteins and the rapidity with which the pools are replenished in children with PEM have not been determined.

Many investigators have proposed that the lower plasma protein concentrations in children with PEM are due to slower rates of synthesis, an adaptive response to poor dietary intakes of protein (Ingenbleek et al. 1975, Smith et al. 1973) and that repletion of the plasma pools in response to nutritional rehabilitation is due to faster rates of synthesis of these proteins in response to the reestablishment of adequate intakes of dietary protein (Dhansay et al. 1991, Ingenbleek et al. 1975, Smith et al. 1973). However, this proposal is not based on actual measurements of the synthesis rates of these proteins but on indirect evidence from human studies. These observations include lower plasma concentrations of RBP, TTR and HDL apoA1 associated with severe PEM (Dhansay et al. 1991, Ingenbleek et al. 1975, Smith et al. 1973), the rapid decrease in plasma concentrations of these proteins in response to decreased protein and energy intakes (Shetty et al. 1979) and the rapid increase in concentrations on refeeding (Ingenbleek et al. 1975, Shetty et al. 1979, Toure et al. 1990). Furthermore, previous work from our laboratory has demonstrated that initial increases in the plasma pools of albumin and transferrin do not occur through an increased rate of synthesis but probably via a reduced rate of catabolism (Morlese et al. 1996 and 1997) during rehabilitation of infected malnourished children. At present, there are no studies in the literature documenting the actual synthesis rates of RBP, TTR and HDL apoA1 and the response to nutritional rehabilitation in the acutely malnourished child.

The present study was undertaken in severely malnourished children to determine whether an increased rate of synthesis is the mechanism responsible for repletion of the plasma pools of the nutrient transport proteins RBP, TTR and HDL apoA1 and the rate at which the pools are repleted during nutritional rehabilitation. With a stable isotope tracer method, the rates of synthesis of RBP, TTR and HDL apoA1 were measured in children with PEM, at three time points during hospitalization: immediately after fluid resuscitation (postadmission d 2); after infections were under control and edema was lost (postadmission d 8); and after the catch-up growth rate had started to plateau and patients had achieved 90% weight for height (~postadmission d 59).

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Subjects.  This study was approved by the Medical Ethics Committee of the University Hospital of the West Indies and the Baylor Affiliates Review Board for Human Subject Research of Baylor College of Medicine. Twenty-two Jamaican children (14 males, 8 females) were enrolled in the study after written informed consent was received from their parents. The physical characteristics of the children at admission are shown in Table 1. None of the children had clinical signs of vitamin A deficiency. The main selection criterion for inclusion in the study was a deficit in body weight of 20%. The weight of the children was measured using a beam balance (Sartorius model F150S, Göttingen, Germany), and length was measured on a horizontally mounted stadiometer (Holtain, Crymych, United Kingdom).

The children were admitted to the metabolic ward of the Tropical Metabolism Research Unit (TMRU) and managed according to a standard treatment protocol (Jackson and Golden 1988). This involved correction of fluid and electrolyte imbalances and administration of broad-spectrum antibiotics (parenteral penicillin, gentamycin and oral metronidazole). After admission, the children were started immediately on a maintenance milk-based diet, which provided 417 kJ energy and 1.2 g·kg¹·d¹ protein with additional supplements of vitamins and trace elements (Table 1) (Morlese et al. 1997) until appetite was restored at ~8 d postadmission. During the catch-up growth phase, the patients were fed a milk-based formula (made energy-dense by the addition of an oil), which provided 625-750 kJ energy and ~3 g protein·kg¹·d¹.

Study design.  The study design has been described in detail previously (Morlese et al. 1996). Briefly, the children were fed the maintenance diet in each study and were studied at three time points. The first isotope infusion was performed after fluid resuscitation when the children were stable (as indicated by blood pressure, heart rate and respiration rate), and they had been on the maintenance diet for 2 d. Study 2 was performed ~8 d postadmission while the children were still on the maintenance diet. At this stage, they had lost edema, their appetite had recovered and their infections were under control as determined by normalization of temperature, respiratory and pulse rates and resolution of clinical features of the infective episode (e.g., cessation of diarrhea and absence of chest crepitations). Study 3 was performed at ~59 d postadmission when the child had fully recovered, that is, after the catch-up growth rate had started to plateau and weight-for-height was 90%. At this point, the child was restarted on the maintenance diet for 3 d before the isotope infusion.

Isotope infusion.  A sterile solution of ²H₃-leucine (Cambridge Isotope Laboratories, Woburn, MA) prepared in 9 g/L of NaCl was infused for 8 h to measure the rates of synthesis of RBP, TTR and HDL apoA1 as previously described (Morlese et al. 1996). Briefly, ~40% of the subject's daily food intake was administered by constant intragastric infusion starting 2 h before the isotope infusion commenced and continued throughout the isotope infusion. After a 2-mL venous blood sample was drawn, ²H₃-leucine was infused nasogastrically at a rate of 26 µmol·kg¹·d¹ for a period of 8 h. Additional 2-mL blood samples were drawn at 2-h intervals throughout the infusion. The same infusion and blood sampling protocol was repeated in the second and third studies.

Sample analyses.  Blood was drawn in prechilled tubes (containing Na₂EDTA and a cocktail of sodium azide, merthiolate and soybean trypsin inhibitor) and immediately centrifuged at 1000 × g for 15 min at 4°C. The plasma was removed and stored at 70°C for later analysis.

Plasma RBP, TTR and HDL apoA1 concentrations were measured by radial immunodiffusion using human RBP, TTR and HDL apoA1 NL RID kits (The Binding Site, San Diego, CA). RBP and TTR were isolated from plasma by sequential immunoprecipitation with anti-human TTR (Behring, Somerville, NJ) and anti-human RBP (Behring, Somerville, NJ) as previously described (Jahoor et al. 1996). Very low density lipoprotein (VLDL)-apoB-100 was separated by ultracentrifugation and isopropanol precipitation as previously described (Jahoor et al. 1994). After the VLDL layer was removed, the HDL fraction of plasma was separated on a 1.21 g/mL of NaBr-EDTA gradient by ultracentrifugation at 450,000 × g and 22°C for 16 h. The RBP and TTR immunoprecipitates and the HDL supernatant were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the RBP and TTR protein from their specific antibodies and to separate the apo A1 moiety from HDL. A pure standard of each protein and low-molecular-weight standards were included in each gel (Jahoor et al. 1996). After staining with Coomassie brilliant blue dye, the bands corresponding to the protein standard were cut out and washed several times. The dried protein precipitates were hydrolyzed in 6 mol/L of HCl at 110°C for 12 h. The amino acids released from the protein were purified by cation exchange chromatography and the tracer/tracee ratio of the protein-derived leucine was determined by negative chemical ionization gas chromatography-mass spectrometry on a Hewlett-Packard 5988A gas chromatography-mass spectrometer (Palo Alto, CA). The amino acids were converted to the n-propyl ester, heptafluorobutyramide derivative, and the leucine tracer/tracee ratio was determined by monitoring ions at m/z 349 to 352.

Calculations and statistics  The fractional synthesis rates (FSR) of RBP, TTR and HDL-apoA1 were calculated with the precursor-product equation: where PEt₂  PEt₁ is the increase in enrichment of RBP (or TTR, HDL apoA1)-bound leucine over the period t₂  t₁ h of the infusion. For HDL apoA1 and TTR, this was t₈  t₄ h and for RBP t₆  t₄ h. E_pl is the plateau enrichment of apoB-100-bound leucine. In this calculation, the plateau enrichment of apoB-100 bound leucine in plasma is assumed to represent the enrichment of the intrahepatic leucine pool from which the RBP (or TTR or HDL apoA1) is synthesized (Jahoor et al. 1994). The steady-state tracer/tracee ratio was obtained by finding the average of the individual tracer/tracee ratio values after the tracer/tracee ratio-time curve reached a plateau. Plateau was defined as previously described (Jahoor et al. 1994).

The absolute intravascular synthesis rate (i.v.ASR) of RBP (or TTR, or HDL apoA1) was estimated as the product of FSR and the intravascular RBP (or TTR, or HDL apoA1) mass: where the intravascular RBP (or TTR, or HDL apoA1) mass is the product of the plasma volume and the plasma concentration of RBP (or TTR, or HDL apoA1). The plasma volume of the acutely ill child was assumed to be 42 mL/kg edema-free body weight (Viart 1976), whereas the value of 50 mL/kg was used for the recovered children (James and Hay 1968, Viart 1976).

The data were analyzed using analysis of variance with repeated measures utilizing the StatView^TM II statistical package (Abacus Concepts, Berkeley, CA) to determine differences between the three studies. When there were significant differences over time, individual studies were compared with univariate postanalysis of variance contrasts. Unpaired t tests were used to determine differences between subjects with edema and those without. Significance of difference was assumed at P < 0.05, and numerical data are expressed as means ± SEM.

	RESULTS

Abstract Introduction Methods Results Discussion References

At admission, 21 of the subjects had evidence of infection based on the presence of one or more of the following: leucocyte count >11 × 10⁹ cells/L, temperature at admission >37 or <35.5°C, positive blood, urine or stool culture. All of the subjects were anemic, 10 were edematous (4 kwashiorkor and 6 marasmic-kwashiorkor) and 12 were marasmic. During study 2, the infections were under control, as indicated by the absence of all signs and symptoms of infection and edematous children had lost their edema. All of the children were severely protein-energy malnourished with a mean weight-for-age of 56 ± 2.1% and a mean weight-for-length of 76 ± 2.0% of expected. These indices of nutritional status were unchanged from study 1 to study 2 but increased significantly by study 3 (Table 1).

The tracer/tracee ratio of leucine bound in VLDL-apoB-100 reached steady state after 4 h of the isotope infusion in all three studies (Fig. 1), and there was a linear increase in the amount of labeled leucine incorporated into plasma RBP (Fig. 1), TTR and HDL apoA1 (Fig. 2) during this period of time. Hence, the FSR of TTR and HDL apoA1 were calculated from the rate of incorporation of labeled leucine into the protein during the last 4 h (4-6 h for RBP) of the isotope infusion. The rate of incorporation of ²H₃-leucine into RBP was linear in all subjects in all studies 6 h of the isotope infusion. However, in a few children, because of the very fast FSR of RBP, the linear incorporation of label into RBP started to plateau toward 8 h of the isotope infusion. Therefore, based on this observation we decided to use the same time points (4 and 6 h) for all subjects to calculate the FSR of RBP.

View larger version (17K): [in this window] [in a new window]   Fig 1. The net tracer/tracee molar ratio (mol/100 mol above baseline) of leucine incorporated into plasma very low density lipoprotein-apoB-100 (VLDL-apoB100) (top panel) and the net tracer/tracee molar ratio of leucine incorporated into plasma retinol-binding protein (bottom panel) during an 8-h intragastric infusion of 2H3-leucine in children with protein-energy malnutrition (PEM) and after recovery from PEM. Each data point represents the mean of 22 subjects. SEM were smaller than the symbols.

View larger version (15K): [in this window] [in a new window]   Fig 2. The net tracer/tracee molar ratio (mol/100 mol above baseline) of leucine incorporated into plasma transthyretin (top panel) and the net tracer/tracee molar ratio of leucine incorporated into plasma high density lipoprotein-apolipoprotein A1 (HDL apoA1) (bottom panel) during an 8-h intragastric infusion of 2H3-leucine in children with protein-energy malnutrition (PEM) and after recovery from PEM. Each data point represents the mean of 22 subjects. SEM were smaller than the symbols.

Retinol-binding protein.  As shown in Figure 3, the mean plasma RBP concentration of the 22 malnourished children was significantly lower (P < 0.05) in study 1 than in study 2 (when infections were under control) or study 3 (when the patients were fully recovered). The plasma RBP concentration in study 2 was not different from that of study 3. There was no difference in the FSR of RBP among the three studies, but the ASR of RBP was significantly lower (P < 0.05) in study 1 than in study 3. The ASR of RBP in study 2 was not different from that of study 3. 

View larger version (19K): [in this window] [in a new window]   Fig 3. Plasma retinol-binding protein (RBP) concentration, fractional synthesis rate (FSR) and intravascular absolute synthesis rate (ASR) in children with protein-energy malnutrition (PEM) at post-admission day 2 (study 1), day 8 (study 2) and day 59 (study 3). Values are means ± SEM of 22 subjects. *Significantly different from study 3 value, P < 0.05. Significantly different from study 2 value, P < 0.05.

When the subjects were divided into edematous and nonedematous groups, the plasma RBP concentration and synthesis rates were not different between the two groups in study 1 or study 2. However, the plasma RBP concentration of the previously nonedematous children was significantly (P < 0.05) lower compared with the edematous group (28.12 ± 1.5 vs. 35.31 ± 5.9 mg/L) at recovery.

Transthyretin.  The plasma TTR concentration was significantly lower (P < 0.05) in study 1 than in studies 2 and 3 (Fig. 4). The plasma TTR concentration in study 2, however, was not different from that of study 3. There was no difference in the FSR of TTR among the three studies, but the ASR of TTR was significantly lower (P < 0.05) in study 1 than in studies 2 and 3, and in study 2 there was no difference in ASR compared to study 3. 

View larger version (19K): [in this window] [in a new window]   Fig 4. Plasma transthyretin (TTR) concentration, fractional synthesis rate (FSR) and intravascular absolute synthesis rate (ASR) in children with protein-energy malnutrition (PEM) at post-admission d 2 (study 1), d 8 (study 2) and d 59 (study 3). Values are means ± SEM of 22 subjects. *Significantly different from study 3 value, P < 0.05. Significantly different from study 2 value, P < 0.05.

When the subjects were divided into edematous and nonedematous groups, the plasma TTR concentration and synthesis rates were not different between the two groups in study 1 or study 2. However, when recovered, the plasma TTR concentration of the nonedematous group was significantly lower (P < 0.05) than that of the edematous group (116.0 ± 7.8 vs. 154.4 ± 13.2 mg/L), and the nonedematous children also had a significantly lower (P < 0.05) ASR of TTR than the children who were previously edematous (3.2 ± 0.4 vs. 4.8 ± 0.7 mg·kg¹·d¹).

High density lipoprotein-apolipoprotein A1.  The plasma HDL apoA1 concentration of the 22 malnourished children was lower in studies 1 and 2 than in study 3 (P < 0.05, Fig. 5). Although the FSR of HDL apoA1 was significantly faster in study 1 than in study 3 (P < 0.05), the ASR of HDL apoA1 was significantly lower in studies 1 and 2 compared with the value at study 3 (P < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig 5. Plasma high density lipoprotein-apoA1 (HDL apoA1) concentration, fractional synthesis rate (FSR) and intravascular absolute synthesis rate (ASR) in children with protein-energy malnutrition (PEM) at postadmission d 2 (study 1), d 8 (study 2) and d 59 (study 3). Values are means ± SEM of 22 subjects. *Significantly different from study 3 value, P < 0.05.

When the subjects were divided into edematous and nonedematous groups, the plasma HDL apoA1 concentration and synthesis rates were not different between the two groups in study 1 or study 2. However, at study 3, the HDL apoA1 concentration and synthesis rates were not different in the edematous compared with nonedematous groups.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

This study was undertaken to determine whether an increased rate of synthesis was the kinetic mechanism responsible for repletion of the pools of three nutrient transport proteins, TTR, RBP and HDL apoA1, and at which rate repletion occurs during nutritional rehabilitation of severely malnourished children. The results show that the repletion of the pools of these different nutrient transport proteins is achieved by the same mechanism, i.e., increased synthesis, but at different rates of induction of the response. The plasma concentration of RBP and TTR increased rapidly to values within the normal range by 8 d postadmission as the amounts of RBP and TTR synthesized increased to values similar to those at recovery. The HDL apoA1 concentration, on the other hand, remained unchanged at 8 d postadmission as the synthesis rate of HDL apoA1 remained significantly lower in study 2 compared to recovery. Additionally, the marasmic children, when recovered, had smaller RBP and TTR pools and synthesized less TTR per unit of time than the recovered children who were previously edematous.

The lower plasma concentrations of RBP, TTR and HDL apoA1 in the severely malnourished children are in agreement with the previous findings of Ingenbleek and Toure (Ingenbleek et al. 1975, Toure et al. 1990). After 8 d of treatment, the plasma concentrations of RBP and TTR increased to values comparable with those at recovery but the plasma HDL apoA1 concentration remained unchanged, suggesting that the repletion of this plasma protein is achieved either through a different mechanism than that of RBP and TTR or at a much slower rate.

The pool size of a plasma protein is determined by the balance between its rates of synthesis and catabolism or loss from the intravascular compartment. Hence, the repletion of a plasma protein pool can be achieved by one of two kinetic mechanisms: an increased rate of synthesis relative to catabolism/intravascular loss or a decreased rate of catabolism/loss relative to synthesis rate. Although in the present study only the rate of synthesis was measured, our results show that the pools of the three nutrient transport proteins were repleted through a similar kinetic mechanism during nutritional rehabilitation. That is, replenishment of the plasma pools were associated with higher synthesis rates. This is in contrast to our previous findings that the plasma pools of albumin and transferrin are replenished by changes in both the rates of catabolism and synthesis (Morlese et al. 1996 and 1997). The initial increase in pool size of these two proteins after 8 d of nutritional rehabilitation was due to a reduced rate of catabolism followed by a marked increase in the rate of synthesis. Taken together, these findings indicate that replenishment of the plasma pools of transport proteins do not occur through identical kinetic mechanisms.

Whereas replenishment of the pools of RBP, TTR and HDL apoA1 occurred through a common mechanism, the rapidity of the repletion of the pools varied. It was clear that the plasma pools of RBP and TTR were repleted by 8 d postadmission but not that of HDL apoA1. This finding in the present study may be due to the fact that RBP and TTR have smaller pools and faster turnover rates compared with HDL apoA1, a protein that has a relatively larger pool and slower turnover rate (Lichtenstein et al. 1990). Hence, the RBP and TTR pools are replenished faster than that of HDL apoA1. Another explanation for the faster replenishment of the RBP and TTR pools may be that these two proteins are physiologically more important than HDL apoA1 for the immediate recovery of the child from malnutrition. This speculation is supported by the fact that low plasma concentrations of RBP and TTR are associated with increased morbidity and mortality in children with severe PEM (Bistrian 1977, Brasseur et al. 1994). Thus failure to quickly replete these plasma proteins could compromise the rate of recovery of the patient.

It is possible that the synthetic rates of these plasma proteins are lower in acute severe PEM due to a shortage of amino acid precursors as a result of a poor antecedent dietary protein intake. This is borne out by the results of Moullac et al. (1992), who reported reduced hepatic RBP and TTR mRNA after rats had consumed a diet providing only 60% of protein requirements for 14 d. Thus during nutritional rehabilitation, as the exogenous supply of amino acids is increased gradually, there is a rapid stimulation of the rate of synthesis of RBP and TTR and hence repletion of the RBP and TTR pools. The findings of a study by Shetty et al. (1979) supports this contention. The RBP and TTR plasma concentrations of a group of obese women subjected to dietary restriction increased to normal values after 4 d of refeeding (Shetty et al. 1979).

When the subjects were divided into edematous and nonedematous groups, there were no differences in the concentration and synthesis rates of the plasma proteins between the groups in studies 1 and 2. However, the recovered kwashiorkor and marasmic-kwashiorkor children had higher plasma RBP and TTR concentrations compared with the recovered marasmic children. This finding is in agreement with our previous finding that edematous children have higher plasma albumin concentrations than those children who were previously marasmic (Morlese et al. 1996). Marasmic children also synthesized less TTR than previously edematous children at recovery. These data support the notion that previously marasmic children have smaller plasma protein pools and synthesize plasma proteins at a slower rate. We speculate that if this is not a direct effect of the postnatal nutritional insult that resulted in severe malnutrition, it may represent an intrauterine programmed effect that confers a survival benefit to the marasmic children, enabling them to adapt more successfully to subsequent nutritional stresses.

One aspect of our results, although not a major finding of our study, generally supports the proposal of Dhansay and Toure that the plasma concentration of HDL apoA1 may be a useful indicator of protein nutritional status (Dhansay et al. 1991, Toure et al. 1990). Unlike RBP and TTR, the plasma concentration of HDL apoA1 does not increase rapidly to a normal value in study 2, a time when there was no improvement in anthropometric measurements.

In conclusion, the findings of this study suggest that the repletion of the nutrient transport protein pools with nutritional rehabilitation is achieved by the same mechanism, i.e., an increased rate of synthesis but at different rates. Repletion of the pools of RBP and TTR, proteins with a quicker turnover rate, is achieved rapidly, whereas repletion of the HDL apoA1 pool is achieved more slowly.

	ACKNOWLEDGMENTS

We are grateful to the nursing staff of the TMRU for their care of the children and to Leslie Loddeke for editorial assistance.

	FOOTNOTES

Manuscript received 15 August 1997. Initial reviews completed 23 September 1997. Revision accepted 31 October 1997.

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