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The Journal of Nutrition Vol. 128 No. 8 August 1998,
pp. 1342-1348
,
Service des Maladies Infectieuses et Tropicales, Service d'Hygiène Hospitalière et * Service d'Endocrinologie et Maladies Métaboliques CHU de Clermont-Ferrand, 63003 Clermont-Ferrand and ** Unité d'Etude du Métabolisme Azoté, INRA Centre de Clermont-Ferrand-Theix, 63122 Ceyrat, France
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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This study was conducted to identify the most rate-limiting amino acids for whole-body protein synthesis in acquired immunodeficiency syndrome (AIDS) patients. We postulated that an essential amino acid that would be rate limiting in AIDS should have a low basal plasma concentration and should remain at a low level during amino acid infusion. Seven male AIDS patients (median age 37 y, CD4 cell count: 76 mm
3) without any clinically active opportunistic infection during the month before the experiment were infused intravenously with a complete amino acid-glucose mixture for 2.5 h. Eight healthy volunteers were used as controls. Before the infusion, the concentrations of most free essential amino acids (methionine, threonine, histidine, isoleucine, leucine and tryptophan) were significantly lower (P < 0.05) in AIDS patients than in controls. Most plasma free essential amino acids increased significantly during infusion. However, the absolute increase above basal levels for threonine, valine, lysine, (P < 0.05) and methionine (P < 0.073) was smaller in AIDS patients than in control subjects. Thus, threonine and possibly methionine may be rate limiting for whole-body protein synthesis in AIDS patients, suggesting that there are selective amino acid requirements in patients with AIDS.
Infection with the human immunodeficiency virus (HIV)5 has a devasting effect on nutritional status. Weight loss, often profound in magnitude, is one of the most universal features of HIV infection, and patients may lose 30-50% of their body mass before succumbing to the disease (Gorbach et al. 1993 Understanding the changes in protein and amino acid metabolism in HIV infection is crucial because they underlie the loss of protein (i.e., of lean tissue). Measurements of amino acid kinetics in AIDS patients have shown the characteristic features of a hypermetabolic response with increased protein turnover (Lieberman et al. 1994 Undernutrition may contribute to the protein wasting in HIV patients because nutritional support seems to have been beneficial (Boulétreau et al. 1995 Subjects.
The study group consisted of seven men aged 30-43 (median 37 y) recruited from the Department of Infectious Diseases at the University Hospital in Clermont-Ferrand. On the bases of the ELISA and Western blot assay, all were HIV seropositive. A clinical history and a physical examination were performed at the time of the study (Table 1). Six patients were classified C3 (lymphocyte count <200) and one C2 (T4 lymphocytes between 200 and 499) according to the criteria of the Centers for Diseases Control and Prevention (1993). Only patients who had been free of any clinically active opportunistic infection for a period of
Experimental procedure.
All studies were performed in a postabsorptive state (12-h overnight fast). At 0800 h, a sampling catheter (Venflon 2, 20G, Viggo, Helsingborg, Sweden) was inserted into a dorsal vein of the left forearm. Another catheter was placed in a contralateral forearm vein and used for infusions. Each experiment consisted of a 150-min period of continuous infusion of the amino acid mixture Primene 5% (1 mL/(kg·h); Clintec Technologies, Velizy-Villacoublay, France). Infusions were performed using a peristaltic pump (Infusomat Secura, Braun Biotrol, Paris). The nitrogen content of the amino acid mixture was 7.5 g/L and the amino acid concentration (g/L) was as follows: L-isoleucine 3.35, L-leucine 5.00, L-valine 3.80, L-lysine 5.50, L-methionine 1.20, L-phenylalanine 2.10, L-threonine 1.85, L-tryptophan 1.00, L-alanine 4.00, L-arginine 4.20, L-aspartic acid 3.00, L-cysteine hydrochloride 1.23, L-glutamic acid 5.00, glycine 2.00, L-histidine 1.90, L-proline 1.50, L-serine 2.00, L-tyrosine 0.45, L-ornithine 1.13 and taurine 0.30. A glucose solution (100 g/L; Meram, Melun, France) was concomitantly administered at a constant rate [1 mL/(kg·h)] via the same catheter by a separate peristaltic pump. Venous blood samples (20 mL) were obtained before ( Assays.
Plasma tryptophan was determined by a fluorometric procedure after conversion to norharman by heating in acid conditions with formaldehyde and ferric chloride (Tesseraud et al. 1992 Statistical analysis.
All data are expressed as means ± SEM. A paired t test was used to compare basal data with data obtained during infusions. A two-way ANOVA ( Plasma cholesterol, albumin, prealbumin, retinol binding protein, triglycerides, and C-reactive protein did not differ between AIDS patients and controls (Table 2). The C-reactive protein was used as a marker to establish the lack of opportunistic infection at the time of the study. By contrast and as expected, the
Loss of lean body mass in HIV patients can result from undernutrition or from disease-induced alterations in metabolism. The lower basal plasma concentrations of most essential (P < 0.05 for threonine, methionine, isoleucine, leucine, histidine and tryptophan) and nonessential (P < 0.05 only for aspartate plus asparagine, glycine and citrulline) free amino acids in patients with AIDS are consistent with a state of protein undernutrition. Indeed, it has been found that reducing protein supply in rats during constant energy intake results in a decrease in the concentrations of most free amino acids in plasma (Grizard et al. 1977
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Sauerwein 1993). Acquired immunodeficiency syndrome (AIDS) is characterized by a predominant loss of lean tissue (Kotler 1985). Multiple factors in different combinations contribute to AIDS related malnutrition and increased host requirements; these include anorexia, malabsorption, abnormal utilization and excretion of nutrients. This is correlated with the severity of the HIV infection and with secondary infections. Malnutrition has a deleterious effect on immune function and thus may potentially accelerate the progression of immune deficiency in HIV infection (Chandra 1991).
, Macallan et al. 1995, Stein et al. 1990). In addition, the degradation of tryptophan via the kynurenine pathway is stimulated (Werner et al. 1988), and sulfur amino acids and glutathione metabolism are altered (Buhl et al. 1989, Eck et al. 1989, Hortin et al. 1994).
, Melchior et al. 1996, Sukkar & Giacosa 1995). Short-term parenteral hyperalimentation enriched with amino acids is capable of reversing net protein catabolism (Macallan et al. 1995, Selberg et al. 1995). However, the amino acid requirements for the replenishment of protein mass in adult humans are not known with certainty (Pellet 1990, Reeds et al. 1994). The aim of this study was to define the amino acids that are most limiting for protein anabolism in AIDS patients on the basis of plasma free amino acid response (Pion 1973, Tontisirin et al. 1974, Zello et al. 1995) to a short-term intravenous infusion of an amino acid-glucose mixture.
SUBJECTS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
1 mo before participation were included. The patients' body weight loss was ~5% [compared with pre-illness body weight (BW)]. Patients with fever (>37.8°C) or diarrhea (defined as increased frequency or liquidity of stools) were excluded. Eight male volunteers, who were HIV negative and clinically well, served as a control group. All patients gave a written informed consent. The study protocol was approved by the local Ethics Committee (Comité Consultatif pour la Protection des Personnes en Recherche Biomédicale pour la Région Auvergne).
View this table:
Table 1.
Characteristics of the acquired immunodeficiency syndrome (AIDS) patients and the control subjects
15 and 5 min), during (15, 30, 60, 90, 120 and 150 min) and after the infusion (15 and 30 min). Samples were collected in heparinized tubes, centrifuged at 4°C for 6 min at 3000 mg and plasma stored at 20°C for subsequent analysis.
). Plasma cyst(e)ine was determined directly by spectrophotometry after coloration with the acid ninhydrin reagent (Malloy et al. 1981). Plasma samples were prepared for analysis of other amino acids by mixing 3 mL plasma with 7 mL of 150 g/L trichloracetic acid containing 0.16 mL thiodiglycol (to prevent methionine oxidation) and 0.75 µmol norleucine (as an internal standard). After storage at 0°C for 1 h and centrifugation at 4000 × g for 30 min at 4°C, the supernatant was passed through a 3-mL cation-exchange column (Dowex AG 50WX8, 100-200 mesh; Bio-Rad, Richmond, CA). Amino acids were eluted from the column with 4 mol/L NH4 OH. The eluate was evaporated to dryness under reduced pressure at 50°C and reconstituted with 3 mL of 0.1 mol/L lithium buffer, pH 2.2. The concentrations of individual amino acids were determined by an automated ion-exchange chromatography apparatus (Biotronic LC 3000, Roucaire, Vélizy, France with BTC 2410 resin), utilizing postcolumn ninhydrin derivatization.
1 acid-glycoprotein and C-reactive protein were measured by immunonephelometry (Beckman, Array System 360, Gagny, France).
= 0.05, main effects: HIV infection and glucose-amino acid infusion) was used to compare results in HIV-infected subjects and controls.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
1 acid glycoprotein was greater (P < 0.05) in AIDS patients than in controls.
View this table:
Table 2.
Routine biochemical variables of the acquired immunodeficiency syndrome (AIDS) patients
and control subjects
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Fig 1.
Plasma insulin, glucose and urea concentrations in patients with acquired immunodeficiency syndrome (AIDS) and control subjects. All variables were measured before (BASAL) and during the combined amino acid-glucose infusion (INFUSED). Values are means ± SEM for eight controls and seven AIDS patients. The insulin values represent one determination at time 15 min (before infusions, i.e., basal state) and the mean of three determinations at times 90, 120 and 150 min during infusions. The glucose and urea plasma values are the mean of two determinations in the basal state, i.e., times 15 and 5 min (before infusion), and five determinations at times 30, 60, 90, 120 and 150 min during infusions. +INFUSED significantly different from BASAL, P < 0.05.
View this table:
Table 3.
Basal plasma free amino acids in control subjects and acquired immunodeficiency syndrome (AIDS) patients
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Fig 2.
Plasma free amino acid concentrations in patients with acquired immunodeficiency syndrome (AIDS) and control subjects. Amino acids were assayed at various times in each group during a basal period (times 15 and 5 min), during the combined amino acid-glucose infusions (indicated by open boxes) and after the infusions (times 165 and 180 min). Values are means ± SEM for eight controls and seven AIDS patients. *Significantly different from controls, P < 0.05. +Significantly different from the values at 15 or 5 min in the same group, P < 0.05.
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Fig 3.
The absolute increase in plasma essential free amino acid concentrations during combined amino acid-glucose infusions in control subjects and acquired immunodeficiency syndrome (AIDS) patients. The plasma free amino acids concentrations were measured at times 15 and 5 min (before infusions) and at times 90, 120 and 150 min during infusions. The absolute increase of each amino acid represents the difference between the mean concentration obtained during and before infusions. Values are means ± SEM for eight controls and seven human immunodeficiency virus (HIV)-infected patients. *Significantly different from controls, P < 0.05. +Significantly different from zero, P < 0.05.
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Fig 4.
The absolute increase of plasma nonessential free amino acid concentrations during combined amino acid-glucose infusions in control subjects and acquired immunodeficiency syndrome (AIDS) patients. The plasma free amino acid concentrations were measured at times 15 and 5 min (before infusions) and at times 90, 120 and 150 min during infusions. The absolute increase of each amino acid represents the difference between the mean concentration obtained during and before infusions. Values are means ± SEM for eight controls and seven AIDS patients. *Significantly different from controls, P < 0.05. +Significantly different from zero, P < 0.05. Glx = Glu + Gln. Asx = Asp + Asn.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). However, the lower concentrations of plasma free amino acids in HIV patients could not be due to protein deprivation because dietary protein assessment (based on an 8-d diet recall) suggests a more than adequate daily protein intake (2.0 ± 0.3 g/kg BW). In addition, no specific energy restriction was detected (daily intake at 222 ± 29 kJ/kg BW) in these subjects. Our study therefore adds evidence to the hypothesis that a state of undernutrition does not exist in stable HIV patients, defined as those free of clinically active opportunistic infections, fever and diarrhea (Sauerwein 1993). However, these adequate intakes did not preclude muscle wasting, and many HIV patients have a history of long-term weight loss.
, Vente et al. 1989). Although HIV infection demonstrates many of the characteristics of a catabolic process, the initial study of protein metabolism in HIV infection using [15N] glycine as a tracer (Stein et al. 1990) found reduced rates of whole-body protein turnover. However, more recent investigations using [13C] leucine have suggested that the rates of protein turnover are high in cachectic AIDS patients (Lieberman et al. 1994, Macallan et al. 1995).
, McLarney et al. 1996, Zello et al. 1995).
, Castellino et al. 1987 and 1992, Fukagawa et al. 1989, Pacy et al. 1988, Tessari et al. 1987). An even greater inhibition of endogenous leucine appearance was also seen when amino acids were combined with glucose and insulin (Bennet et al. 1990, Castellino et al. 1987, Flakoll et al. 1989, Fukagawa et al. 1989, Heslin et al. 1992, Tauveron et al. 1995, Tessari et al. 1987). The splanchnic bed is the major site of the disposal of intravenously administrated amino acids (Gelfand et al. 1986). Studies in animals suggest that intravenous amino acids are catabolized preferentially by the liver and thus reduce the amounts of amino acids arising from proteolysis (Mortimore et al. 1987). Liver protein synthesis may also be stimulated (Tauveron et al. 1994). Amino acid deposition in skeletal muscle cannot be ruled out because an increase in muscle protein synthesis by hyperaminoacidemia has been reported in both healthy volunteers (Bennet et al. 1989) and animals (Mosoni et al. 1993, Watt et al. 1992). Interestingly, it has been shown that the acute anabolic response to intravenous amino acid infusion was normal in HIV-infected subjects (Macallan et al. 1995, Selberg et al. 1995).
27% compared with controls). In addition, the absolute increase in plasma free threonine after infusions (although significant, P < 0.05) was very modest in HIV-infected subjects, representing only 52% of the increase in controls. Based on the curve describing blood free threonine in response to the consumption of graded levels of threonine (Pion 1973, Tontisirin et al. 1974), our results suggest that HIV-infected patients have a selective deficiency in threonine. Such a deficiency has also been demonstrated recently in septic rats by using amino acid balance methodologies (Arnal et al. 1995). This selective threonine deficiency could arise from an activation of the catabolism of threonine and/or synthesis of threonine-rich proteins.
). To explain the decrease in plasma free methionine, we hypothesize that in patients with AIDS, there is a concomitant activation of the metabolism of methionine to cyst(e)ine. In contrast to previous studies (Hortin et al. 1994), cyst(e)ine depletion was not observed in our experiments although abnormal kinetics were recorded during infusions. We also hypothesize that more sulfur amino acids are needed in patients with AIDS to meet their requirements for glutathione (
-glutamylcysteinylglycine) synthesis. Cysteine is both a precursor and a regulator of glutathione synthesis. HIV-infected patients are glutathione deficient (López Galera et al. 1996), presumably as a result of an enhanced utilization due to activation of lymphocytes and cell-mediated cytotoxic function and protection against oxidative damage. Similar mechanisms have been proposed as explanations for the increased sulfur amino acid requirements during sepsis in rats (Malmezat et al. 1998).
). This may contribute to the neurologic symptoms often associated with the HIV infection. Although the basal plasma tryptophan concentrations were significantly lower in HIV patients than in controls, this amino acid increased similarly after infusion in both groups in our experiment and thus could not be considered rate limiting as was the case for threonine and methionine.
for a review). In this study, the utilization of glycine for glutathione synthesis was also presumably enhanced (see above). Alternatively, the appearance rate of glycine may have been decreased, first, because glycine synthesis from serine may have been decreased as a result of an increased utilization of serine for cysteine synthesis and second, because glycine can also be synthesized from threonine. We speculate that changes in threonine metabolism led to a decrease in glycine appearance. A drastic decrease in plasma free glycine has been observed in patients in response to multiple traumas (Grimble 1990).
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FOOTNOTES |
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Manuscript received 14 July 1997. Initial reviews completed 13 October 1997. Revision accepted 13 April 1998.
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ACKNOWLEDGMENTS |
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The authors thank Clintec Technologies for providing Primene 5% and Anthony Kee for improving the manuscript.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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effect of insulin and amino acids.
Am. J. Physiol.
1992;
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