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,,3
*
Department of Nutritional Sciences and
Paediatrics, University of Toronto, Toronto, Canada M5S 3E2;
The Research Institute, The Hospital for Sick Children, Toronto, Canada M5G 1X8;
**
School of Dietetics and Human Nutrition, McGill University, Montreal, Canada H9X 3V9; and
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada T6G 2P5
3To whom correspondence should be addressed. E-mail: paul.pencharz{at}sickkids.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSEstimation of isotope kineticsRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • histidine • whole-body protein metabolism • indicator amino acid oxidation • healthy adults
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSEstimation of isotope kineticsRESULTSDISCUSSIONLITERATURE CITED |
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), despite controversy regarding its essentiality. It was found that histidine was not required for the maintenance of nitrogen balance in short-term (6- to 8-d) studies (2
,3
); however, it was not clear whether histidine was required to maintain nitrogen equilibrium in long-term (
30-d) studies (4
–6
). Kopple and Swenseid (4
) found that nitrogen balance was negative in healthy adults and uremic patients when diets were devoid of histidine for 25–36 d; but in the study of Wixom et al. (5
), nitrogen balance remained positive in one subject receiving parenteral nutrition without histidine for 27 d. In Cho’s study (6
) nitrogen balance was 0 or positive in subjects consuming a histidine-deficient diet for 56 d; however, upon reintroduction of histidine into the diet, nitrogen balance rapidly became more positive. To date, whether histidine is an indispensable amino acid in healthy adults has not been clearly resolved.
The maintenance of large histidine pools in the form of hemoglobin (Hb)4
and carnosine is a possible reason for the difficulty in establishing the essentiality of histidine. When a histidine-deficient diet was consumed for a prolonged period, a decrease in Hb, in conjunction with a rise in serum iron, was observed (4
,6
). These data suggest that during limited dietary supply, histidine pools may be maintained through the release of histidine from the degradation of Hb (4
), and/or the reduction in Hb synthesis (7
). Histidine may also be released from carnosine (ß-alanyl-L-histidine), a dipeptide present in large quantities in skeletal muscle (8
). Carnosine concentrations in muscle and/or olfactory bulb were reduced in rats (9
–12
), chickens (13
), cockerels (14
) and dogs (15
) consuming a histidine-deficient diet. Free carnosine concentration in muscle tissue in normal adults is 20–180 mg/100 g (16
). Thus, during consumption of a histidine-deficient diet, the amount of carnosine in muscle tissue is theoretically sufficient to provide enough histidine to maintain nitrogen balance in older children and adults for several weeks (8
), but not in infants due to the limited amount of the enzyme carnosinase (17
,18
). Decreased oxidation and degradation of histidine (9
,19
,20
) is another possible adaptive response in the body to maintain histidine adequacy during consumption of a histidine-deficient diet.
Further knowledge of the effect of histidine deficiency in human adults is required to determine whether histidine is an indispensable amino acid. Stable isotope techniques allow us to understand more about protein and amino acid metabolism (21
,22
). To our knowledge, no one has previously used isotope kinetics to study histidine deficiency in humans. Therefore, this study was designed to investigate the effects of a histidine-free diet on protein metabolism assessed by the indicator amino acid oxidation (IAAO). Nitrogen balance and metabolism, and biochemical indices were also used to assess the effect of histidine deficiency on protein metabolism of healthy adults.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSEstimation of isotope kineticsRESULTSDISCUSSIONLITERATURE CITED |
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Healthy adults (3 men and 1 woman) participated in the study on an outpatient basis at the Clinical Investigation Unit of the Hospital for Sick Children (HSC), Toronto, Canada. None of the subjects had a history of histidine and/or carnosine metabolism disorders, unusual dietary practices, recent weight loss, chronic disease, endocrine disorders or medication use before or during the study. The purpose of the study and the potential risks associated with the protocol were fully explained to each subject. Written informed consent was obtained for the study, which was approved by the Research Ethics Board of the HSC. The subjects received financial compensation for their participation. They were encouraged to maintain their usual physical activities throughout the study period.
Experimental design.
The 52-d study consisted of a 4-d complete amino acid diet followed by a 48-d histidine-free diet. Every 4 d, an IAAO study was performed using L-[1-13C]phenylalanine as the indicator. Urine samples (24-h) were collected the day before each IAAO study for measurement of histidine, ß-alanine, 3-methylhistidine (3-MH), creatinine, urea nitrogen and total urinary nitrogen excretion. Albumin, transferrin, complete blood count (CBC), ferritin and plasma amino acids were measured at baseline (d 0) and on d 24 and 48. CBC and ferritin were also determined on d 78 ± 2 and 108 ± 2 during which self-selected diets were consumed. Subjects were weighed on each IAAO study day to ensure accurate prescription of diets and isotopes, and to confirm weight maintenance throughout the study. Body composition including lean body mass (LBM) and fat-free mass (FFM) was determined on the first and last IAAO study day. LBM was calculated from reactance and resistance (23
) measured by bioelectrical impedance analysis (BIA, model 101A; RJL Systems, Detroit, MI). FFM was calculated from density derived from the four skinfold thickness (triceps, biceps, subscapular and suprailiac crest) (24
) measured by a skinfold caliper (British Indicator, St. Albans, UK).
Whole-body protein turnover was also measured in one subject (WK) using the [15N]-urea end-product method with oral [15N]glycine (25
) every 4 d during the study period. Baseline and 48-h urine was collected for this particular subject before and after providing a single dose (20 mg) of [15N]glycine (98%, Cambridge Isotope, Woburn, MA). [15N]glycine was consumed in the early morning after collecting baseline urine 2 d before each IAAO study day. This subject also consumed a histidine-repletion diet for 4 d after the histidine-free diet, and two additional IAAO, plus one [15N]glycine study were performed during this repletion period.
Dietary intake and experimental diet.
The diet provided throughout the 52 d was a liquid formula diet with protein-free cookies as previously developed for amino acid kinetic studies (26
). The protein content was supplied as a crystalline L-amino acid mixture based on the amino acid composition of egg protein at a level of 1 g/(kg · d). For the histidine-free diet, histidine was replaced with glycine to keep the same amount of nitrogen in egg protein. Energy requirement was based on each subject’s resting metabolic rate after a 12-h overnight fast, as determined by continuous, open-circuit indirect calorimetry (2900 Computerized Energy Measurement System-Paramagnetic; Sensormedics, Yorba Linda, CA) and multiplied by an activity factor of 1.7–2.0 depending on activity levels of each participant. The main source of energy in the diet was supplied from a modified liquid formula diet containing no amino nitrogen (Protein-Free Powder, Product 80056, Mead Johnson, Evansville, IN; Tang and Koolaid, Kraft, Don Mills, Toronto, Canada) with the remaining energy coming from protein-free cookies. Overall, the proportions of energy in the experimental diet were 53% carbohydrate, 37% fat and 10% protein. The diet was portioned into four isocaloric and isonitrogenous meals that were consumed daily at 0800, 1200, 1600 and 2000 h. No other food or beverages were consumed during the study period except water, clear tea or clear decaffeinated coffee. A daily multivitamin supplement including folic acid (Centrum, Whitehall-Robins, Mississauga, Canada) was also provided to each subject 2 wk before the study and continually until the end of the study. Dietary fiber was provided as 24 g psyllium hydrophilic mucilloid (Life, Shoppers Drug Mart Pharmaprix, Toronto, Canada) to all subjects. Compliance to the experimental diet protocol was monitored by the maintenance of a low level of urinary histidine in each subject participating in the study.
The diet consumed on the IAAO study day was the same as that described above except the amount of phenylalanine was reduced to 15 mg/(kg · d) (to account for the amount of L-[1-13C]phenylalanine administered via the tracer). This level of phenylalanine intake had previously been determined to meet the requirements of 95% of adult males when tyrosine was presented in excess [40 mg/(kg · d)] (26
). Glycine intake was increased on the study day to maintain the same intake of nitrogen. On the IAAO study day, the experimental diet was administered as 7 isocaloric, isonitrogenous hourly meals each representing 10% of the daily requirement. The subjects consumed portions of the meal hourly to ensure a metabolic steady state in the fed condition (27
,28
). Subjects consumed the hourly meals beginning 2 h before infusion of the isotope to reach a baseline plateau (28
). Subjects were also provided the rest of the diet (30% of the daily requirement) to consume at home, after the completion of the IAAO study.
Tracer protocol.
Two tracers, NaH13CO3 (99%) and L-[1-13C]phenylalanine (99%) (Cambridge Isotope), were used in this study. Isotopic and optical purity of L-[1-13C]phenylalanine was verified by the manufacturer of the isotopes using chemical ionization gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance. The enrichment and enantiomeric purity of the L-[1-13C]phenylalanine tracer were reconfirmed by GC-MS of the n-propyl, heptofluorobutyramide derivative (29
) using a chiral column (ChirasilVal, R symbol; Alltech Associates, Deerfield, IL). The measured fractional molar abundance of L-[1-13C]phenylalanine was 97.5%. This value was used in the calculation of phenylalanine turnover. The tracers were prepared in deionized water and stored at –20°C until used. All tracers were given orally with the experimental diet. A bolus dose of 2.07 µmol NaH13CO3/kg, and 3.99 µmol L-[1-13C]phenylalanine/kg was given to prime the bicarbonate and phenylalanine pools, respectively. In addition to the priming dose, the subjects were given 7.99 µmol L-[1-13C]phenylalanine/kg hourly throughout the remaining 5 h of the study.
Sample collection.
On the IAAO study days, three baseline urine and expired breath samples were collected 15, 30 and 45 min before starting the isotope protocol. Five urine and breath samples were collected at isotopic steady state every 30 min during the period 150–270 min after initiation of the isotope protocol. This isotope protocol had been shown to achieve isotopic steady state in 2 h after the start of the L-[1-13C]phenylalanine isotope procedure (30
). The same pattern also was seen in the present study as demonstrated in Figure 1
. Breath samples were collected in disposable Haldane Priestley tubes (Venoject; Terumo Medical, Elkton, MD) using a collection mechanism that permits the removal of dead-space air and stored at room temperature pending analysis. Urine samples were kept at -20°C until analyzed for L-[1-13C]phenylalanine enrichment. Expired CO2 production rate was measured using an indirect calorimeter (2900 Computerized Energy Measurement System; Sensormedics, Yorba Linda, CA) after 4 h of consuming the experimental diet on each IAAO study day.
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Complete 24- or 48-h urine was collected in a container containing 40 mL of 3.4 mol/L hydrochloric acid. The urine volume was measured and an aliquot was stored at -20°C. Completeness of the urine collections was verified by the constancy of daily creatinine excretion.
Analytical procedures.
Expired 13CO2 enrichment was measured by a continuous flow isotope ratio mass spectrometer (model 20/20, PDZ Europa, Cheshire, UK), and was expressed as atom % excess against a reference standard of compressed CO2 gas. Urinary L-[13C]phenylalanine enrichment was measured by the modified method of Patterson et al. (29
) using negative chemical ionization GC-MS with a chirasil Val-D fused-silica capillary column. Briefly, a 1-mL urine sample was deproteinized and acidified with 500 µL of 2.5 mol/L trichloroacetic acid and centrifuged at 7000 x g. Amino acids were separated from the supernatant using cation exchange columns (Dowex 50W-X8, 100–200 mesh, H+ form, Bio Rad, Hercules, CA). Eluate from the column was freeze-dried (Freezone 12 L; Labconco, Kansas City, MO) before amino acids were derivatized to their N,O-heptafluorobutyryl n-propyl ester derivatives. Using methane-negative chemical ionization GC-MS (Hewlett-Packard 5890 series; GC; Hewlett-Packard 5988A MS system, Mississauga, Canada), selected-ion chromatograms were obtained by monitoring [M - HF] ions at m/z 383 for L-phenylalanine and m/z 384 for L-[1-13C]phenylalanine. Isotope enrichment in mol % excess was calculated from peak area ratios at isotopic steady state and baseline.
Total urinary nitrogen content was determined in diluted urine samples (100 µL urine:10 mL deionized water) by pyrochemiluminescence using an ANTEX 7000 nitrogen/sulfur analyzer (Mandel Scientific, Houston, TX) (31
). Urinary urea was measured by a colorimetric method on the Vitros BUN/UREA slides, and urinary creatinine was assayed by the two-point rate test on the Vitros CREA slides (Vitros 950, Ortho-Clinical Diagnostic, Rochester, NY). Urinary urea nitrogen was isolated, after removal of ammonia, using urease and a modification of the Conway diffusion method (32
). The [15N] enrichment of urinary urea was determined by combustion using an elemental analyzer (ANCA, Europa) at 1000°C, interfaced with a continuous flow isotope the ratio mass spectrometer (Model 20/20, PDZ Europa). The 15N-14N ratios (m/z) 28:29:30 were measured against a calibrated urea standard.
Plasma free amino acids were separated using a cation exchange column, as mentioned earlier, using norleucine as an internal standard. Plasma and urinary amino acid concentrations were measured by reversed-phase HPLC (Dionex Summit HPLC System, Dionex, Sunnyvale, CA; operated under HPLC pump model P580A LPG and UV/VIS Detector UVD 170S) of their phenylisothiocyanate derivatives (adapted from PicoTag; Waters, Milford, MA) (33
). The areas under the amino acid peaks were integrated using Chromeleon Software Version 6.2 (Dionex).
Plasma albumin was determined by a colorimetric method on the Vitros ALB slides (Vitros 950). Plasma transferrin was measured by immunonephelometry on the Behring Nephelometer II (Dade Behring Marbury, Marburg, Germany). Serum ferritin was assayed by Heterogenous Sandwich Magnetic Separation Assay on the Technicon Immono1 System (Bayler, Tarrytown, NY); CBC was analyzed by the Coulter method (Coulter GEN-S-System, Beckman Coulter, Miami, FL).
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Estimation of isotope kinetics |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSEstimation of isotope kineticsRESULTSDISCUSSIONLITERATURE CITED |
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Phenylalanine kinetics were calculated according to the stochastic model of Matthews et al. (34
). Isotopic steady state in the metabolic pool was represented by a plateau in free L-[1-13C]phenylalanine in urine and 13CO2 in breath. The difference between mean breath 13CO2 enrichment or urinary L-[1-13C]phenylalanine of the baseline and plateau samples was used to determine atom or mol % excess above baseline at isotopic steady state.
Phenylalanine flux and oxidation [µmol/(kg · h)] were calculated from the measured dilution of the L-[1-13C]phenylalanine into the body amino acid pool when isotopic equilibrium was reached (34
). The rate of 13CO2 released by phenylalanine tracer oxidation [F13CO2, in µmol13CO2/(kg · h)] was calculated from the 13CO2 enrichment of expired air at isotopic steady state (34
). The factor of 0.82 was used to account for the 13CO2 retained in the body in the fed state, as a result of bicarbonate fixation (35
). The rate of phenylalanine oxidation [µmol/(kg · h)] was calculated from F13CO2 and urinary phenylalanine enrichment (34
,36
).
Protein turnover with a single dose of [15N] glycine.
The single-dose [15N]glycine study was analyzed using the cumulative excretion model described by Waterlow and co-workers (37
), which is based on the assumption that the proportion of isotope excreted in the end product should be the same as the proportion of the nitrogen flux excreted in that end product. The urea end product pool was used in this study. The total flux of amino nitrogen (QN) was calculated from the following equation:
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where d is the amount of 15N administered, E is the amount of urea-N excreted and ec is the cumulative excretion of 15N-urea at plateau.
The pathways of exit of nitrogen from the whole-body amino nitrogen pool are by synthesis to protein and by oxidation of the amino acid followed by excretion of the nitrogen as urea in the urine. Therefore, the whole-body protein synthesis and breakdown can be calculated by the following equation:
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where I is total nitrogen intake, B is whole-body protein breakdown, S is whole body protein synthesis and E is total urinary excretion.
Statistical analysis.
One-way ANOVA with repeated measures was used to determine the effects of study days on F13CO2, phenylalanine kinetics, plasma and urinary amino acid concentrations, plasma protein and hematologic concentrations. When warranted, post-hoc analysis was performed with Duncan’s multiple range test. Linear regression was performed to evaluate the pattern of change in body weight and 15N-glycine kinetics during consumption of the histidine-free diet. Regression models (linear, quadratic and linear regression crossover) were fit to data to determine the relation that best described the response of the dependent variables (phenylalanine flux and oxidation) to a duration of consuming the histidine-free diet. The comparisons for LBM and FFM determined on the first and last IAAO study day were tested by paired two-tailed t tests. All statistical analyses were performed using the SAS program (version 8.0, SAS Institute, Cary, NC). Data are presented as means ± SEM, and differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSEstimation of isotope kineticsRESULTSDISCUSSIONLITERATURE CITED |
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. Two of four subjects felt hungry and lost some weight during the first few days of consuming the complete amino acid diet. Therefore, the total energy intake of those two subjects was increased from 1.8 to 2.0 times resting metabolic rate and was held constant thereafter. There was an effect of study days on body weight (P < 0.001). Linear regression for individual subjects showed a small but gradual decrease in body weight in three of four subjects (slope significantly different from zero) during consumption of the histidine-free diet (Fig. 2
). However, there were no differences in estimates of LBM (P = 0.89) and FFM (P = 0.63) determined on the first and last IAAO study days (data not shown). All subjects felt healthy throughout the study except one (HR) who had a mild cold and complained of nausea during d 19–22.
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The urinary excretion of urea and total nitrogen did not change during the 48 d of consuming the histidine-free diet (Fig. 3
). Total urinary excretion was 143.9 ± 32.6 mg N/(kg · d), with day-to-day variations ranging from 12 to 22%. Fecal and miscellaneous nitrogen losses were not measured directly in this study. Using a value for fecal nitrogen loss of 12 mg/(kg · d) and miscellaneous nitrogen loss of 8 mg/(kg · d) (1
), nitrogen balance during the histidine-free diet was -3.9 ± 30.7 mg N/(kg · d) [ranging from -24.6 to 16.4 mg N/(kg · d)]. These nitrogen balances did not differ from zero, and there was no significant slope of change in nitrogen balance in individual subjects.
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), an index of muscle mass, and 3-MH (Fig. 4B
), an index of muscle catabolism, were not affected by consumption of the histidine-free diet. Daily urinary creatinine excretion, indicating the completeness of 24-h urine collection, was 0.24 ± 0.02 mmol/(kg · d) with a CV of 9.8%.
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The plasma histidine level declined markedly with ingestion of the histidine-free diet. At the end of the study (d 48), plasma histidine had fallen to 38.4 ± 8.8% of the concentration at the onset of the study (Table 2
). Within the first 3 d of initiation of the histidine-free diet, urinary histidine fell markedly by 51.5 ± 14.6%. Urinary histidine levels continued to decrease slowly, and by the end of the histidine-free diet period, the level was only 18.5 ± 8.3% of the initial value (Fig. 4
A). In one subject who received the histidine repletion diet for 4 d after consuming the histidine-free diet, a marked rise in urinary histidine excretion to the initial level was observed (initial = 617 µmol/d; repletion = 802 µmol/d). Urinary ß-alanine excretion was not affected by the histidine-free diet. However, the smallest subject (WK) excreted more ß-alanine during consumption of the histidine-free diet (P = 0.05 by linear regression) (Fig. 4
C). Other amino acid concentrations were also measured; however, no particular changes were observed and the results are not presented.
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Figure 5
depicts the pattern in rate of 13CO2 released by phenylalanine tracer oxidation (F13CO2) during the 48 d of consuming the histidine-free diet. There was some variation, but no significant change was detected in F13CO2 measured every 4 d during consumption of the histidine-free diet. However F13CO2 at baseline (d 0), when all subjects were receiving a complete amino acid diet, was higher than the F13CO2 obtained during the consumption of the histidine-free diet (P = 0.004). In one subject, who consumed the histidine repletion diet for 4 d after the histidine-free diet, the F13CO2 dropped by 18% compared with the mean F13CO2 of this subject during the histidine-free diet (depletion = 0.371 ± 0.042; repletion = 0.306 ± 0.005), indicating increased whole-body protein synthesis.
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. A two-phase linear regression crossover model was the best fit and indicated that there was a slow, linear decline in phenylalanine flux during the first 30 d of the histidine-free diet, with no change in phenylalanine flux after d 30 (Fig. 6
A). At the end of study period (d 48), phenylalanine flux had decreased by 28.7% compared with baseline. Phenylalanine oxidation dropped dramatically (48.4%) during the first 8 d of consuming the histidine-free diet, after which the values remained fairly constant (Fig. 6
B).
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[15N]urea enrichment after administration of [15N]glycine measured in one subject (WK) increased significantly between d 0 and 47 (P = 0.003), resulting in a lower calculated flux of amino nitrogen (P = 0.005). Linear declines in calculated whole-body protein synthesis (P = 0.016) and breakdown (P = 0.005) also occurred during the histidine depletion period. By the end of the study, whole-body protein turnover had decreased by 24% compared with the baseline value. Although these results are in a single subject, they parallel and support the change in phenylalanine flux.
Plasma protein and hematologic variables.
With continued ingestion of the histidine-deficient diet, plasma albumin and transferrin decreased in all 4 subjects (P = 0.04), although they remained within normal ranges (Table 2
). Concentrations of Hb (P = 0.003), hematocrit (Hct, P = 0.007) and RBC (P = 0.04) also decreased during the histidine depletion period (Table 3
). These three variables fell by 10% after 24 d of consuming the histidine-free diet, and no further decrease had occurred at the end of the study (d 48). Mean cell hemoglobin (MCH) dropped significantly on d 48 compared with d 0 (P = 0.026). Calculated mean cell volume (MCV) of subjects did not change during the 48 d of histidine depletion (P = 0.18). Serum ferritin rose 41.5 ± 26.7% by d 24 (P = 0.0003) compared with the initial level, and no further increase was observed at the end of histidine depletion. In light of the changes observed, blood was taken every week from the last 2 subjects during the histidine-free diet period, and the same pattern of results was observed. After consuming a self-selected diet for 1 and 2 mo after the histidine-free diet, Hb, Hct and RBC counts had slowly increased, but were still lower than the initial values.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSEstimation of isotope kineticsRESULTSDISCUSSIONLITERATURE CITED |
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). Nitrogen balance was not different from zero. This result is in agreement with the study of Wixom et al. (5
) who found that nitrogen balance was slightly positive in one subject during 27 d of total parenteral nutrition devoid of histidine. However, this finding is in contrast to the study of Kopple and Swendseid (4
), which showed a negative nitrogen balance after 5–36 d of consuming a histidine depletion diet. The difference in nitrogen balance results may be due to the amount of total protein and/or amino acid provided. Kopple and Swendseid provided only 0.63 g protein/(kg · d) (4
), which is lower than the total protein of 0.93 and 1.0 g/(kg · d) provided by Wixom et al. (5
) and in the present study, respectively.
L-[1-13C]phenylalanine was used as an indicator to monitor the effect of the histidine-free diet on whole-body protein kinetics. Phenylalanine flux decreased from baseline during the histidine-free diet period, indicating an overall deceleration of protein turnover. This situation may be considered to be an accommodation of the human body in response to a prolonged period of lacking exogenous histidine (39
) because irreversible loss of physiologic function (decrease in whole-body protein synthesis and breakdown) was observed. This finding is in agreement with the study of Kino and Okumura (40
) who reported that absolute whole-body protein synthesis and breakdown decreased in chicks fed a histidine-free diet compared with a control diet. The rapid decrease in F13CO2 and phenylalanine oxidation during the first 4–8 d of consuming the histidine-free diet indicates a physiologic response of humans to utilize amino acids efficiently to maintain protein balance. The reductions in phenylalanine flux (28.7%) and oxidation (48.4%) in this study are similar to the findings of Lariviere et al. (41
) who found that leucine appearance and oxidation decreased by 17 and 55% in normal and well-controlled diabetic subjects after adaptation to a protein-free diet. Hoffer et al. (42
) also reported a reduction in postprandial amino acid oxidation and an increase in net protein utilization after dietary protein restriction. The lack of change in urinary nitrogen and urea in the face of change in protein turnover is consistent with a successful accommodation to dietary histidine deprivation. The decrease in phenylalanine oxidation was more pronounced than the decrease in flux, resulting in a positive net protein balance.
A limitation of this study is that there was no control group of subjects fed a complete amino acid diet (including histidine) for the same period (48 d). However, it is unlikely that decreases in phenylalanine flux and oxidation would occur when a complete amino acid diet is consumed. In our earlier study determining the phenylalanine requirement of men (43
), we found no change in phenylalanine flux during 9 d of consuming a complete amino acid diet, interrupted on d 3, 6 and 9 for assessment of the responses to the phenylalanine doses. More recently, Kurpad et al. (44
) extended their 24-h indicator amino acid balance study to determine the lysine requirement during a 3-wk period of consuming an amino acid diet, with variation in the lysine intake. There were no differences in leucine flux, oxidation (either 12-h fast and 12-h fed) and 24-h leucine balance at the end of 7 and 21 d of consuming the amino acid diet. Although the subjects in the present study consumed the histidine-free diet for a longer period (48 d), the greatest decline in phenylalanine oxidation, followed by a slower drop, occurred within the first week of consuming the experimental diet. Therefore, the present results are probably not related to the amino acid diet itself, but to a lack of histidine. In addition, one subject in the present study had an 18% decrease in F13CO2 when a histidine repletion diet was consumed for 4 d after the histidine-free diet. Again, an acute response in oxidation was observed when a complete amino acid diet was reintroduced. Therefore, we contend that the changes in phenyalanine kinetics in the present study are related to the lack of histidine in the experimental diet as opposed to the nature of the diet itself.
This is the first application of the IAAO method using phenylalanine kinetics in response to depletion of histidine, which has not been clearly defined as an indispensable amino acid in human adults. We were aware that there may be some limitations to using phenylalanine kinetics to represent whole-body protein metabolism. Therefore, measurement of whole-body protein turnover using oral [15N]glycine with urea as an end product was conducted in one subject. Similar linear declines in whole-body protein turnover for [15N]glycine and L-[1-13C]phenylalanine were observed. Total flux of amino nitrogen decreased by 24% by the end of the study (d 47), which is comparable to the 29% reduction in phenylalanine flux on d 48. This suggests that the effect of the histidine-free diet on phenylalanine kinetics is an acceptably accurate representation of whole-body protein turnover kinetics. Based on the principle of IAAO, the change in F13CO2 (main outcome measurement of IAAO method) is affected mainly by the intake of the test amino acid. If the intake of the test amino acid meets the requirement for protein synthesis, there will be no change in F13CO2 (22
,36
). The issue of whether the test amino acid is dispensable or indispensable may not be a limitation of using IAAO method.
An adaptive decrease in protein turnover during the absence of histidine was supported by corresponding significant decreases in albumin, transferrin and Hb concentrations. The decrease in albumin concentration in this study is in agreement with the study of Kopple and Swendseid (4
), but not with those of Wixom et al. (5
) and Cho et al. (6
) who found no change in albumin during 27 and 56 d of histidine depletion, respectively. No previous study has reported transferrin level during a histidine-deficient diet. Limited Hb synthesis, in which iron is not incorporated normally into Hb, is suggested by the decrease in erythrocyte indices, which occurred in conjunction with a rise in serum ferritin. Although MCH decreased, there was no change in MCV (Table 3
), indicating that new RBC have less Hb per cell. The numerical increase (NS) of Hb, HCT and RBC counts, and the abrupt, significant decline (P = 0.005) in serum ferritin when subjects resumed self-selected diets after the histidine depletion period, strengthen the suggestion that dietary histidine is necessary to maintain the concentrations of these blood indices. The slow and nonsignificant increase in Hb and RBC concentrations during the histidine repletion period may be due to the long half-life of RBC (4 mo) (45
).
The reduction in Hb resulting from the removal of histidine from the diet in the present study was 16.3 g/L (decreased from 164.8 to 148.5 g/L; Table 3
). Because Hb contains 8% histidine (46
) and the mean blood volume in normal adults is 5 L (45
), the change in Hb accounts for 6.5 g histidine over 24 d. This is equivalent to 3.6 mg/(kg · d) of histidine (based on the average body weight of 75 kg). Therefore, during a limited dietary supply of histidine, 3.6 mg of histidine/kg body is used for hemoglobin synthesis each day. By way of comparison, the present FAO/WHO/UNU (1
) estimate of the histidine requirement in adults is 8–12 mg/(kg · d). Therefore, Hb synthesis may comprise 40% of daily dietary histidine needs.
During histidine deficiency, carnosine may serve as a source of histidine (9
,13
,47
) due to its metabolism to ß-alanine and histidine (48
). In the present study, we measured the urinary concentration of ß-alanine as a marker of carnosine breakdown. We observed a trend toward increased excretion of ß-alanine only in the smallest subject during the histidine-free diet period (Fig. 5
C). However, ß-alanine may not be a sensitive enough index of carnosine catabolism due to possible further metabolism of ß-alanine (49
). ß-Alanine was shown to be rapidly and extensively oxidized to carbon dioxide and acetic acid in rats injected with 14C-ß-alanine (49
). Evidence that carnosine can be a reservoir in muscle for histidine, which can be utilized in the body during histidine deficiency, has been found in rats (9
–12
,47
,50
). Cianciaruso et al. (15
) reported a reduction in muscle carnosine in adult dogs fed a histidine-free diet for 59 d. However, when they studied net release or uptake of histidine and carnosine in kidneys of dogs, they found that there was no adaptive increase in carnosine utilization during histidine deficiency in either short ( 6 d) or long-term (57 d) studies (51
). Thus, it is possible that decreased formation of carnosine occurred in dogs during histidine deficiency. A study in horses showed that changes in muscle carnosine concentration appeared to be influenced by ß-alanine more than histidine bioavailability (52
). No reports exist on the status of muscle carnosine during a histidine-deficient diet in humans. Therefore, the issue of whether carnosine is a store for histidine that can be utilized in the human body during histidine deficiency must be further investigated.
Two other possible mechanisms that may help explain a reduction in or lack of dietary supply of histidine are decreased oxidation and degradation of histidine, and in vivo synthesis of histidine. When the amino acid supply is altered, the uptake into liver and the rate of catabolism may be changed to maintain the stability of the amino acid pools (53
). Findings from animal studies of histidine degradation during a histidine deficiency diet are paradoxical. Kang Lee and Harper (20
) observed a fall in the hepatic histidase and urocanase activities when histidine was removed from the diet of rats. However, Torres et al. (54
) found that the activity of histidase increased (accompanied by an increase in Hal-mRNA abundance) when rats were fed a histidine-free diet. Currently there are no studies in the literature that address the issue of whether histidine deficiency down-regulates histidine metabolism. Evidence exists that histidine is synthesized in the human body. Sheng et al. (55
) reported an incorporation of 15N into both the 
-amino nitrogen and the imidazole ring of histidine in plasma and urine of an adult man receiving histidine-free total parenteral nutrition who orally ingested 15NH4Cl. From that study, the high enrichment of 15N in histidine from fecal bacterial cells suggested possible bacterial histidine synthesis. Another study showed that the 15N from urea was incorporated into all amino acids except lysine and threonine in both plasma and muscle protein in normal and catabolic subjects, but it was not incorporated into histidine in uremic patients (56
). Although histidine may be synthesized in the human body, the data in the present study indicate that if synthesis occurs, it is not sufficient to maintain the histidine pool.
In conclusion, when using the IAAO method, our data suggest that there was a rapid ( 1 wk) decrease in amino acid oxidation, followed by a gradual decrease in protein turnover, and that by 3–4 wk, there was a substantial decrease in plasma protein and hematologic concentrations, indicating possibly detrimental adaptation (accommodation) during consumption of the histidine-free diet. Therefore, although histidine deficiency may not affect nitrogen equilibrium if the total protein intake is higher than the current recommendation (0.75 g/kg), the other changes in protein metabolism support the view that histidine is an indispensable amino acid in healthy adults. Hence, the definition of an indispensable amino acid should not be restricted to nitrogen equilibrium but should take into account the level of the amino acid that optimizes protein synthesis, amino acid flux and other functional protein parameters. The extensive metabolic accommodation, together with decreases in Hb, albumin and transferrin during histidine depletion leaves unresolved the issue of whether histidine is a dietary essential amino acid.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by grants MT 10321 of the Canadian Institutes of Health Research. W.K. was supported by the Nestle Nutrition Scholarship, Switzerland, Connaught Scholarship, University of Toronto and RESTRACOMP, The Research Institute, The Hospital for Sick Children, Toronto, Canada.
4 Abbreviations used: CBC, complete blood count; FFM, fat-free mass; GC-MS, gas chromatography-mass spectrometry; Hct, hematocrit; Hb, hemoglobin; HSC, Hospital for Sick Children; IAAO, indicator amino acid oxidation; LBM, lean body mass; MCH, mean cell hemoglobin; MCV, mean cell volume; 3MH, 3-methylhistidine.
Manuscript received 2 July 2002. Initial review completed 24 July 2002. Revision accepted 28 August 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSEstimation of isotope kineticsRESULTSDISCUSSIONLITERATURE CITED |
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