1 Department of Nephrology, Istituto G. Gaslini, Genova, Italy, 2 Division of Renal Medicine and Baxter Novum, Department of Clinical Science, Karolinska Institutet, Stockholm, Sweden and 3 Department of Clinical Chemistry II, Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden
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Abstract |
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Methods. Twelve apparently well-nourished chronically uraemic children (five females) aged a mean of 9.4±4.8 (range 1.717.7) years and 13 age-matched normal children were studied. Venous blood and muscle samples for AA analyses were taken simultaneously after an overnight fast.
Results. The intracellular AA patterns in the three cellular compartments were qualitatively similar, but the absolute intracellular concentrations were higher in muscle than in PMN, which had higher values than in RBC. The AA patterns in plasma, RBC, PMN, and muscle in the uraemic children have many similarities; typical features being low branched-chain AA (BCAA), tyrosine, and serine concentrations and variably high concentrations of some non-essential AA. Among the individual AA, there were only few correlations between their concentrations in the three cell compartments.
Conclusions. The lack of correlation between the concentrations in RBC, PMN, and muscle for most of the AA indicates that there is no close association in the same subject between individual free AA concentrations in various types of cells, presumably because of differences in metabolism and function. Consequently, one should be cautious in assuming that AA concentrations, determined in RBC or PMN, reflect the concentrations in muscle cells. Therefore, these preliminary observations do not support the hypothesis that RBC and PMN AA analysis can be considered as a suitable alternative to muscle AA determination.
Keywords: amino acids; leukocytes; muscle; plasma; red blood cells; uraemic children
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Introduction |
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However, the clinical significance of these findings remains unknown, as plasma AA levels can be affected by many factors. Thus, the AA pattern tends to reflect recent protein intake rather than body composition or protein mass. Hence, the use of plasma AA concentrations for the evaluation of body nutrition has been of limited value [2].
Free intracellular AA are the immediate precursors of protein synthesis and their intracellular distribution is one of the major factors in regulating protein metabolism. Determination of free intracellular AA can, therefore, provide useful information about the total pool of each free AA and also about protein metabolism. This is particularly interesting in children since, in early life, the protein synthesis is maximally stimulated.
Intracellular free AA has been studied mostly in skeletal muscle, which contains the largest pool of free AA in the body [3]. Samples of muscle tissue are generally obtained by needle biopsy, an invasive and sometimes uncomfortable procedure, which limits its utilization in children.
Polymorphonuclear granulocytes (PMN) may offer an alternative cell model in which all major metabolic pathways are present [4]. PMN have been used to study the intracellular metabolism of free AA in relation to nutritional factors and in some pathological states [5]. Abnormal AA levels in PMN, which resemble those described in muscle in CRF, have mainly been reported in uraemic children [6].
The intracellular concentrations of AA in PMN are thought to be similar to those found in muscle [6]. However, there are no studies reported in which the intracellular AA pools in PMN and muscle were taken from the same subject at the same time.
Red blood cells (RBC) contain a large proportion of the free AA in blood, the intra-erythrocyte pool of free AA being actively involved in the inter-organ transport of AA [7]. The RBC posses no nuclei, mitochondria, ribosomes, or other organelles and, therefore, cannot synthesize protein. However, numerous AA transport systems have been found in human RBC similar to those in other cells and the AA profile in RBC has been reported as abnormal in patients with CRF [8].
In this study we determined at the same time the AA concentrations in plasma, RBC, PMN, and muscle in normal children and children with end-stage renal disease (ESRD). Our aims were to evaluate whether these concentrations are similar in various compartments and the extent to which they are affected by uraemia.
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Material and methods |
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The uraemic children not yet on dialysis were prescribed a diet with a protein content of 75% and an energy content of 100% of the recommended daily allowance (RDA) for statural age. The children on CAPD were prescribed a diet with a protein and energy content that was 100% of the RDA for statural age. The protein and energy intakes were estimated from dietary recall records that were taken over a 3-day period by a dietician (Table 1). The protein intake in non-dialysed children was also estimated from the 24-h urea excretion, according to Maroni et al. [9] (Table 1
).
The control group comprised 13 children (three girls) having a mean age of 9.1±3.9 (range 2.816.5) years, undergoing elective surgery for hernia correction or uretero-pelvic junction stenosis. They were all in good health, apart from the minor disabilities that required elective surgery, and had no signs of metabolic or renal disease.
Venous blood and muscle samples for AA analyses were taken simultaneously after an overnight fast, in patients and in controls. The muscle biopsy specimens were taken from the rectus abdomini muscle during peritoneal catheter insertion in the ESRF children, during elective surgery for hernia in the children on CAPD and during elective surgery for hernia or uretero-pelvic junction stenosis correction in controls.
The protocol of the study was approved by the Ethics Committee of the G. Gaslini Institute, Genoa, Italy and parent's informed consent was obtained.
Analytical procedures
Serum electrolytes and some routine biochemical factors were evaluated by routine methods. A heparinized blood sample was centrifuged at 4000 r.p.m. for 10 min at +4°C to obtain plasma that was then deproteinized with sulphosalicylic acid and centrifuged. The supernatant was stored at -70°C, pending analyses of AA.
The muscle specimens were carefully and rapidly dissected to remove visible fat and connective tissue. They were repeatedly weighed on a Cahn electromagnetic balance and the initial wet weight was calculated by extrapolating the weight curve to zero time. Immediately afterwards, each specimen was frozen in liquid nitrogen. The frozen material was placed in sodium-free glass tubes, which had been rinsed with nitric acid (1 mol/l), freeze-dried, and re-weighed. The dried, fat-extracted specimen was powdered in an agate mortar and carefully dissected under a magnifying glass to remove remaining flakes of connective tissue. About 2.5 mg of the powder for chloride analysis was dried at 80°C for 30 min and re-weighed. This procedure reduced the water content by approximately 5%. Chloride was extracted, using 1 M nitric acid, and determined by electrometric titration, as described earlier [10]. The true dry-weight of the remaining powder was calculated as 95% of the weight observed after powdering at room temperature and humidity.
Alkali-soluble protein (ASP), that is, non-collagen protein, and DNA were determined in 34 mg of the powder, after precipitation with 4% sulphosalicylic acid. The precipitate was incubated for 1 h in KOH (0.3 mol/l) and ASP was determined in an aliquot, using the Lowry method [11]. DNA analysis of the residues was based on the Schmidt and Tannhauser technique [12]. For DNA extraction, the precipitate was hydrolysed by adding 0.25 ml perchloric acid (1 mol/l) and incubated for 1 h at 70°C. The tube was weighed again to obtain a dilution volume for DNA. DNA was estimated by the diphenylamide reaction [12]. The calculations of extra- and intracellular water contents and intracellular AA concentrations in muscle, based on the chloride method, have been described earlier [10]. Total, extra- and intracellular water, fat, DNA, and ASP are expressed per kg of fat-free solids (FFS). The ASP/DNA ratio is presented as an indicator of the amount of cell protein per cell unit.
For measurement of RBC AA, white cells, and platelets were carefully removed and 1 g of packed red cells was rapidly haemolysed by adding 1.0 ml of 1% Saponin (Sigma, St Louis, MO, USA). The sample was then extracted with 0.3 ml 50% sulphosalicylic acid (SSA), mixed and centrifuged at 1700 g for 20 min at 4°C. The supernatant was filtered using a 0.45 µm HA filter (Millipore), and frozen at -70°C pending analysis. We calculated the intracellular AA concentrations in RBC by taking the water content as 66% of RBC weight in all samples, as described by Flügel-Link et al. [13].
The granulocytes were separated from blood by gradient centrifugation on mono-poly resolving medium (M.PRM). After separation, the cells were washed in Ca2+- and Mg2+-free Hank's balanced salt solution, and the red cells lysed by adding distilled water. After centrifugation, the pellet was suspended in 0.5 ml 0.16 M potassium chloride. Apart from centrifugation, all subsequent procedures were performed with the sample kept in crushed ice. A combination of fresh protease inhibitors was added to the M.PRM, Hank's solution, distilled water and potassium chloride [14]. The mixture of protease inhibitors consisted of leupeptin (1 µM/l), pepstatin (1 µM/l), phenylmethylsulfonyl fluoride (200 µM/l), and EDTA (100 M/l). The cell suspension was lysed by three cycles of freezing and thawing. The cells were frozen at -80°C for 15 min and thawed at 4°C for 60 min. The suspension was deproteinized by adding SSA (7 mg of SSA/ml of suspension). DNA analysis was based on the Schmidt and Tannhauser technique [12]. The intracellular water in PMN correlated with the DNA content that is, intracellular water µl=1.93±0.055 DNA µg (P=0.0001), as described by Metcoff et al. [6]. This relationship was used to calculate cell water, as minimal differences have been reported in direct measurements of PMN water between several uraemic patients and controls.
Free AA were analysed in the supernatants after sulphosalicylic acid precipitation by reversed phase HPLC (Beckman Instruments, Fullerton, CA, USA), using pre-column derivatization with orthophaldialdehyde and an internal standard (homoserine), as described earlier [14]. Taurine, alanine, and 1-methylhistidine (lMHIS) and 3-methylhistidine (3MHIS), respectively, co-eluted in the muscle and in some plasma samples. The combined concentrations of these pairs were used to calculate the sum of AA.
Statistical analysis
As AA concentrations in the four compartments were not normally distributed the differences between the groups were determined with the non-parametric MannWhitney test. Spearman's correlation was used to calculate the relation between AA concentrations and several variables. P<0.05 was considered significant.
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Results |
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The protein intake in the uraemic children, estimated from the dietary recall records, was on average 1.4 g/kg BW/day; the intake estimated from the 24-h urea excretion was in close agreement (Table 1). Their estimated energy intake was, on average, 87% of RDA and the energy intake from the dialysis fluid was negligible (Table 1
).
The weekly renal and peritoneal creatinine clearances in the CAPD children are reported in Table 2.
In the uraemic children, serum levels of total proteins, albumin, transferrin, pseudo-cholinesterase, and serum bicarbonate were about the same as in the controls (Table 2). The muscle ASP contents and ASP/DNA ratios were significantly lower in the uraemic children than in the controls (Table 3
). Total water, extra- and intracellular water in muscle were similar (Table 3
).
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RBC
The RBC concentrations of isoleucine, tyrosine, valine, arginine, and glutamic acid and the valine/glycine and tyrosine/phenylalanine ratios were significantly lower in patients than in controls. The RBC levels of citrulline, 1MHIS+3MHIS and the glycine/serine ratios were significantly increased (Table 4).
PMN
The intracellular concentrations of methionine, phenylalanine, tyrosine, and valine in the PMN were significantly lower than in the controls as were the valine/glycine ratios. Citrulline levels and the glycine/serine ratio were higher and serine lower in PMN of uraemic children (Table 4).
Muscle
The intracellular concentrations of isoleucine, leucine, tyrosine, valine, serine, and the valine/glycine ratio in muscle were significantly lower in the patients than in the controls and the citrulline concentration and the glycine/serine ratio were significantly higher (Table 4).
Gradients
In the uraemic children, the RBC/plasma concentration gradients of glycine and 1MHIS+3MHIS were significantly lower than in the controls (Table 5). The PMN/plasma concentration gradients of methionine, valine, asparagine, glycine, serine, and taurine+alanine were also significantly lower as were the muscle/plasma concentration gradients of isoleucine and phenylalanine.
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Correlations
The data from controls and patients have been plotted separately to assess correlations between individual AA in plasma, RBC, PMN, and muscle and between individual AA and nutritional parameters by univariate analysis. Therefore, we reported the correlations found in each group in Table 6.
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Discussion |
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The overall renal and peritoneal function in the CAPD children was almost comparable with the ESRD children group. The comparison of the AA levels between CAPD vs ESRD showed significant differences only for lysine which was reduced in plasma; asparagine, valine, and taurine+alanine were also reduced in RBC and glutamate and tyrosine were increased in PMN. Therefore, we concluded that ESRD and CAPD patients could be grouped together for the analysis of the AA values.
The uraemic children were considered according to traditional techniques of nutritional assessment to be well-nourished. However, ASP and the ASP/DNA ratio in muscle, which is an accurate quantitative index for assessing the muscle protein content as it reflects the amount of protein per cell nucleus, were lower than in the controls, suggesting that they might have had some degree of subclinical protein malnutrition.
Moreover, in three of the patients in CAPD serum albumin levels were below 30 g/l. However, in ESRD patients serum albumin is a questionable marker of nutritional status [15] as albumin concentration may be decreased by a variety of non-nutritional factors such as fluid overload, urinary and peritoneal fluid albumin losses and inflammation. The absence of correlation between BMI SDS and protein and energy intake expressed as per cent of RDA suggests that other factors, i.e. inflammation, may have a key role in the nutritional status as suggested by Stenvinkel et al. [16].
The wide variations in the ages of the controls and of the patients, implying that the children were at different stages of development, growth rate, and endocrine status may have affected the results. Indeed, we found significant correlations between age and plasma glutamine (P<0.01), plasma isoleucine (P<0.05), plasma leucine (P<0.05), muscle glycine (P<0.05), and muscle threonine (P<0.05).
The results of our study confirm that the free AA concentrations of the various AA in plasma, RBC, PMN, and muscle bear little relation to the average composition of tissue proteins, the AA pattern of dietary protein or the requirements of EAA [17]. The intracellular AA patterns in the three cellular compartments were qualitatively similar, but the absolute intracellular concentrations (Table 4) and the intra- and extracellular gradients (Table 5
), which varied considerably among the AA, were higher in muscle than in PMN, which had higher values than in RBC, presumably reflecting different levels of metabolic activity and differences in membrane transport characteristics.
Intracellular AA concentrations are related to protein synthesis and breakdown, AA transport (uptake and release) and metabolism of AA by oxidation, and other metabolic pathways. Obviously, the balance between these processes differs considerably among the individual AA, thus explaining the large variations in these concentrations under relatively steady conditions, as in the post-absorptive state [18].
The results on free intracellular AA concentrations in RBC, PMN, and muscle are in general agreement with previous publications in normal children and adults [3,6,8,19,20].
It appears that the characteristic AA patterns observed in plasma and muscle tissue of uraemic children are also present in RBC and PMN. Typical features being low branched-chain amino acid (BCAA), tyrosine and serine concentrations, and variably high concentrations of some NEAA.
The low BCAA levels can be considered an early sign of subclinical protein malnutrition, as reflected by a low muscle ASP/DNA ratio.
Given the relative insensitivity of the conventional nutritional assessment techniques used in this study, it is possible that the use of more accurate nutritional markers might reveal more significant correlations between intracellular concentrations of free AA and nutritional indexes. Indeed the presence of significant correlations between muscle ASP/DNA ratio and intracellular concentrations of some free AA support such hypothesis (Table 6). One aim of the present study was to evaluate to what extent the concentrations of AA in plasma, RBC, PMN, and muscle are associated in the same person. To analyse this, we determined using univariate analysis for each AA the correlation between the concentration in each compartment with that in the other compartments in controls and patients respectively.
Among the individual AA, we found only few significant correlations between their concentrations in the three cell compartments (RBC, PMN, muscle) both in controls and uraemic children (Table 6).
The lack of correlation between the concentrations of most of the AA in RBC, PMN, and muscle indicates that there is no close association in the same subject between individual free AA concentrations in various types of cells, presumably because they differ with respect to metabolism and function. Consequently, one should be cautious in assuming that in individual patients, AA concentrations, determined in PMN, reflect the concentrations in muscle cells, as has been suggested [6], although in groups of subjects, the general AA patterns may be similar.
In conclusion, the determination of free AA in RBC and PMN should be seen as a complementary tool in order to add further insight into the nature of the AA and protein metabolism disturbances occurring in uraemia. In particular PMN AA analysis, not requiring an invasive and uncomfortable procedures as muscle biopsy, appears to be a useful cell model to detect and monitor AA abnormalities in uraemia. However, these preliminary observations do not seem to support the hypothesis that RBC or PMN AA analysis can be considered as a suitable alternative to muscle AA determination.
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Notes |
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References |
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