Phenylalanine Turnover Research Articles

An Acute Reduction in Habitual Protein Intake Attenuates Post Exercise Anabolism and May Bias Oxidation-Derived Protein Requirements in Resistance Trained Men.

Protein recommendations for resistance-trained athletes are generally lower than their habitual intakes. Excess protein consumption increases the capacity to oxidize amino acids, which can attenuate post-exercise anabolism and may impact protein requirements determined by stable isotope techniques predicated on amino acid tracer oxidation. We aimed to determine the impact of an acute (5d) reduction in dietary protein intake on post-exercise anabolism in high habitual consumers using the indicator amino acid oxidation (IAAO) technique. Resistance trained men [n = 5; 25 ± 7 y; 73.0 ± 5.7 kg; 9.9 ± 2.9% body fat; 2.69 ± 0.38 g·kg−1·d−1 habitual protein intake) consumed a high (H; 2.2 g·kg−1·d−1) and moderate (M; 1.2 g·kg−1·d−1) protein diet while training every other day. During the High protein phase, participants consumed a 2d controlled diet prior to determining whole body phenylalanine turnover, net balance (NB), and 13CO2 excretion (F13CO2) after exercise via oral [13C]phenylalanine. During the Moderate phase, participants consumed 2.2 g protein·kg−1·d−1 for 2d prior to consuming 1.2 g protein·kg−1·d−1 for 5d. Phenylalanine metabolism was measured on days 1, 3, and 5 (M1, M3, and M5, respectively) of the moderate intake. F13CO2, the primary outcome for IAAO, was ~72 and ~55% greater on the 1st day (M1, P < 0.05) and the third day of the moderate protein diet (M3, P = 0.07), respectively, compared to the High protein trial. Compared to the High protein trial, NB was ~25% lower on the 1st day (M1, P < 0.01) and 15% lower on the third day of the moderate protein diet (M3, P = 0.09). High habitual protein consumption may bias protein requirements determined by traditional IAAO methods that use only a 2d pre-trial controlled diet. Post-exercise whole body anabolism is attenuated following a reduction in protein intake in resistance trained men and may require ~3–5d to adapt. This trial is registered at clinicaltrials.gov as NCT03845569.

Longitudinal Changes in Amino Acid (AA) Turnover in Pregnancy in Women with Normal Glucose Tolerance (NGT) and GDM—Relationship to Fetal Growth

GDM have decreased glucose insulin sensitivity vs. NGT. However, there are less data on the impact of GDM on AA metabolism or relationship to fetal growth. Ten NGT and 8 GDM (≥ 1 abnormal value, 3 hour OGTT) were evaluated at 18-22 and 32-36 weeks for 2 potent amino acid secretogogues, Phenylalanine (PHE) and Leucine (LEU). PHE and LEU turnover was estimated (D5 PHE and 13C LEU) under basal and euglycemic hyperinsulinemic clamp conditions. Neonatal body composition was estimated using anthropometrics. There were no significant difference in maternal age, race, education, tobacco use, weight or body composition between GDM and NGT at 18-22 weeks. The was a higher family hx of diabetes in GDM (p=0.05). Basal LEU but not PHE turnover increased over time and was greater in GDM, Table. Glucose disposal decreased over time and was lower in GDM. During the clamp LEU but not PHE turnover increased over time and was greater in GDM. Clamp PHE turnover (µmol/kg/hour) at 18-22 weeks correlated with neonatal lean mass (r=0.48, p=0.048) and there was a trend for fat mass (r=0.47, p=0.058). Omitting 1 outlier there remained a trend for PHE turnover and neonatal lean mass (r=0.43, p=0.09). Leu turnover increased over time and was greater in GDM vs. NGT. In contrast, there were no significant changes in PHE turnover over time or between groups but PHE turnover in early pregnancy was related to fetal lean mass.Metabolic ResultsNGT (n=10)GDM (n=8)ANOVABasal Leucine turnover (umol/kg*ffm/hr)12-16 weeks129.8 ± 19.8155.6 ± 20.1p=0.00532-36 weeks134.1 ± 20.8157.0 ± 22.9pgroup = 0.001Leucine turnover (umol/kg*ffm/hr)12-16 weeks108.5 ± 18.5126.8 ± 20.8p=0.0232-36 weeks123.0 ± 19.8136.2 ± 21.8pgroup = 0.03mean ± SDpgroup difference between NGT &amp; GDM Disclosure P. Catalano: None. L. Presley: None. S. Hauguel de Mouzon: None. S. Kalhan: None.

Increased Protein Requirements in Female Athletes after Variable-Intensity Exercise.

Protein requirements are primarily studied in the context of resistance or endurance exercise with little research devoted to variable-intensity intermittent exercise characteristic of many team sports. Further, female populations are underrepresented in dietary sports science studies. We aimed to determine a dietary protein requirement in active females performing variable-intensity intermittent exercise using the indicator amino acid oxidation (IAAO) method. We hypothesized that these requirements would be greater than current IAAO-derived estimates in nonactive adult males. Six females (21.2 ± 0.8 yr, 68.8 ± 4.1 kg, 47.1 ± 1.2 mL O2·kg·min; mean ± SE) completed five to seven metabolic trials during the luteal phase of the menstrual cycle. Participants performed a modified Loughborough Intermittent Shuttle Test before consuming eight hourly mixed meals providing the test protein intake (0.2-2.66 g·kg·d), 6 g·kg·d CHO and sufficient energy for resting and exercise-induced energy expenditure. Protein was provided as crystalline amino acid modeling egg protein with [C]phenylalanine as the indicator amino acid. Phenylalanine turnover (Q) was determined from urinary [C]phenylalanine enrichment. Breath CO2 excretion (FCO2) was analyzed using mixed effects biphase linear regression with the breakpoint and upper 95% confidence interval approximating the estimated average requirement and recommended dietary allowance, respectively. Protein intake had no effect on Q (68.7 ± 7.3 μmol·kg·h; mean ± SE). FCO2 displayed a robust biphase response (R = 0.66) with an estimated average requirement of 1.41 g·kg·d and recommended dietary allowance of 1.71 g·kg·d. The protein requirement estimate of 1.41 and 1.71 g·kg·d for females performing variable-intensity intermittent exercise is greater than the IAAO-derived estimates of adult males (0.93 and 1.2 g·kg·d) and at the upper range of the American College of Sports Medicine athlete recommendations (1.2-2.0 g·kg·d).

Fish oil positively regulates anabolic signalling alongside an increase in whole-body gluconeogenesis in ageing skeletal muscle

Fish oil, containing mainly long-chain n-3 polyunsaturated fatty acids (LCn-3PUFA), has been found to acutely stimulate protein synthesis and insulin-mediated glucose metabolism. However, the underlying mechanism and more prolonged effect of fish oil during ageing remain to be determined. Fish oil (EPAX6000; 49.6 % eicosapentaenoic acid, 50.4 % docosahexaenoic acid) or control oil (60 % olive, 40 % soy) supplementation was delivered, via chocolate-derived sweets, to rats for 8 weeks. Throughout the study, food intake and body weight were recorded and body composition was investigated using EchoMRI. During the last 40 min of a 6 h infusion, with labelled dextrose ([U-(13)C]glucose) and amino acids ([1-(13)C]phenylalanine), blood samples were collected to assess glucose and phenylalanine kinetics. Soleus and longissimus dorsi muscles were extracted for protein and mRNA analyses. Fish oil had no effect on food intake or body composition. An increased whole-body glucose turnover, mainly accounted for via an increase in endogenous glucose production, was observed with fish oil feeding. No effects on whole-body phenylalanine turnover were observed. In longissimus dorsi, fish oil augmented the phosphorylation of phosphoinositide 3-kinase (PI3K)([Tyr458]) (P = 0.04) and 70 kDa ribosomal protein S6 kinase (p70s6k)([Thr389]) (P = 0.04). There were no differences in protein kinase B (Akt)([Ser473]), mammalian target of rapamycin (mTOR)([Ser2448]), protein phosphatase 2A (PP2A) 56 kDa regulatory B subunit γ (PP2A-B56-γ), forkhead box containing proteins O-subclass 3a (FOX03a)([Ser253]) or inflammatory markers (Interleukin-6, Interleukin-1 β, tumour necrosis factor-α, and cyclooxygenase-2). Our data suggest that the fish oil may stimulate endogenous glucose production and increase anabolic signalling in ageing rats.

Chronic inflammation in the elderly is associated with increased tryptophan catabolism and altered phenylalanine turnover: Relevance for neuropsychiatric morbidity

Chronic inflammation in the elderly is associated with increased tryptophan catabolism and altered phenylalanine turnover: Relevance for neuropsychiatric morbidity

Chronic Immune Stimulation Correlates with Reduced Phenylalanine Turnover

Neuropsychiatric symptoms like mood changes and depression are common in patients with chronic inflammatory disorders such as infections, autoimmune diseases or cancer. The pathogenesis of these symptoms is still unclear. Pro-inflammatory stimuli interfere not only with the neural circuits and neurotransmitters of the serotonergic, but also with those of the adrenergic system. The pro-inflammatory cytokine interferon-gamma stimulates the biosynthesis of 5,6,7,8-tetrahydrobiopterin (BH4), which is cofactor for several aromatic amino acid monooxygenases and thus is strongly involved in the biosynthesis of the neurotransmitter serotonin and the catecholamines dopamine, epinephrine (adrenaline) and norepinephrine (noradrenaline). In macrophages, interferon-gamma also triggers the high output of reactive oxygen species, which can destroy the oxidation-labile BH4. Recent data suggest that oxidative loss of BH4 in chronic inflammatory conditions can reduce the biosynthesis of catecholamines, which may relate to disturbed adrenergic neurotransmitter pathways in patients.

Dose–response effects of free fatty acids on amino acid metabolism and ureagenesis

Free fatty acids (FFAs) are important fuels and have vital protein-sparing effects, particularly during conditions of metabolic stress and fasting. However, it is uncertain whether these beneficial effects are evident throughout the physiological range or only occur at very high FFA concentrations. It is also unclear whether secondary alterations in hormone levels and ketogenesis play a role. We therefore aimed at describing dose-response relationships between amino acid metabolism and circulating FFA concentrations at clamped hormone levels. Eight healthy men were studied on four occasions (6 h basal, 2 h glucose clamp). Endogenous lipolysis was blocked with acipimox and Intralipid was infused at varying rates (0, 3, 6 or 12 microL kg(-1) min(-1)) to obtain four different levels of circulating FFAs. Endogenous growth hormone, insulin and glucagon secretion was blocked by somatostatin (300 microg h(-1)) and replaced exogenously. 15N-phenylalanine, 2H4-tyrosine and 13C-urea were infused continuously to assess protein turnover and ureagenesis. We obtained four distinct levels of FFA concentrations ranging from 0.03 to 2.1 mmol L(-1) and 3-hydroxybutyrate concentrations from 10 to 360 micromol L(-1). Whole-body phenylalanine turnover and phenylalanine-to-tyrosine degradation decreased with increasing FFA levels as did insulin-stimulated forearm fluxes of phenylalanine. Phenylalanine, tyrosine and urea concentrations also decreased progressively, whereas urea turnover was unperturbed. Circulating FFAs decrease amino acid concentrations and inhibit whole-body phenylalanine fluxes and phenylalanine-to-tyrosine conversion. Our data cover FFA concentrations from 0 to 2 mmol L(-1) and indicate that FFAs exert their protein conserving effects in the upper physiological range (>1.5 mmol L(-1)).

Effects of Cortisol on Carbohydrate, Lipid, and Protein Metabolism: Studies of Acute Cortisol Withdrawal in Adrenocortical Failure

Cortisol is an important catabolic hormone, but little is known about the metabolic effects of acute cortisol deficiency. The objective of the study was to test whether clinical symptoms of weight loss, fatigue, and hypoglycemia could be explained by altered energy expenditure, protein metabolism, and insulin sensitivity during cortisol withdrawal in adrenocortical failure. We studied seven women after 24-h cortisol withdrawal and during replacement control during a 3-h basal period and a 3-h glucose clamp. Cortisol withdrawal generated cortisol levels close to zero, a 10% decrease in basal energy expenditure, increased TSH and T(3) levels, and increased glucose oxidation. Whole-body glucose and phenylalanine turnover were unaltered, but forearm phenylalanine turnover was increased. During the clamp glucose, infusion rates rose by 70%, glucose oxidation rates increased, and endogenous glucose production decreased. Urinary urea excretion decreased by 40% over the 6-h study period. Cortisol withdrawal increased insulin sensitivity in terms of increased glucose oxidation and decreased endogenous glucose production; this may induce hypoglycemia in adrenocortical failure. Energy expenditure and urea loss decreased, indicating that weight and muscle loss in Addison's disease is caused by other mechanisms, such as decreased appetite. Increased muscle protein breakdown may amplify the loss of muscle protein.

Basal muscle intracellular amino acid kinetics in women and men

Sexual dimorphism in skeletal muscle mass is apparent, with men having more muscle mass and larger individual muscle cells. However, no sex-based differences have been detected in blood forearm phenylalanine turnover, although whole body leucine oxidation has been reported to be greater in men than in women. We hypothesized that sex differences in intracellular amino acid turnover may account for these discrepancies, with men having a higher intracellular turnover than women. We studied young, healthy women (women, n = 8) and men (men, n = 10) following an overnight fast. Phenylalanine, leucine, and alanine muscle intracellular kinetics were assessed using stable isotope methodologies, femoral arteriovenous blood sampling, and muscle biopsies. Muscle intracellular amino acid kinetics were reported relative to both leg volume and lean leg mass because of sex differences in leg volume and in muscle and fat distribution. When expressed per leg volume (nmol.min(-1).100 ml leg volume(-1)), phenylalanine net balance (women: -16 +/- 4, men: -31 +/- 5), release from proteolysis in the blood (women: 46 +/- 9, men: 75 +/- 10) and intracellular availability (women: 149 +/- 23, men: 241 +/- 35), and alanine production, utilization, and intracellular availability were higher in men (P < 0.05). However, when the kinetic parameters were normalized per unit of lean leg mass, all differences disappeared. Muscle fractional synthetic rate was also not different between women and men. We conclude that there are no sex-based differences in basal muscle intracellular amino acid turnover when the data are normalized by lean mass. It remains to be determined if there are sex differences in intracellular amino acid metabolism following anabolic or catabolic stimuli.

In vivo rabbit hindquarter model for assessment of regional burn hypermetabolism.

Severe burn injury evokes hypermetabolism and muscle wasting, despite nominally adequate nutrition. Although there is much information on whole organism and isolated tissue metabolism after burn injury, data examining regional burn hypermetabolism in vivo are lacking. Using surgically implanted (general anesthesia) regional vascular catheters and primed constant infusion of l-[1-(13)C]phenylalanine tracer, we have determined in vivo burn-induced alterations in rabbit hindquarter protein and energy metabolism. Burn injury evokes increased whole body resting energy expenditure and phenylalanine turnover, accompanied by significantly increased hindquarter proteolysis, creating a negative protein balance in burned rabbit hindquarter. Hindquarter oxygen consumption showed an increase after burn injury, but it did not reach statistical significance. Burn-induced changes in hindquarter protein turnover account for approximately one-third of the whole animal hypermetabolism. This model offers a system for regional manipulation of postburn hypermetabolism.

Mechanisms of postprandial protein accretion in human skeletal muscle. Insight from leucine and phenylalanine forearm kinetics.

The relative role of protein synthesis and degradation in determining postprandial net protein deposition in human muscle is not known. To this aim, we studied forearm leucine and phenylalanine turnover by combining the arteriovenous catheterization with tracer infusions, before and following a 4 h administration of a mixed meal in normal volunteers. Forearm amino acid kinetics were assessed in both whole blood and plasma. Fasting forearm protein degradation exceeded synthesis (P < 0.01) using either tracer, indicating net muscle protein loss. The net negative forearm protein balance was quantitatively similar in whole blood and in plasma. After the meal, forearm proteolysis was suppressed (P < 0.05- < 0.03), while forearm protein synthesis was stimulated (P < 0.05- < 0.01). However, stimulation of protein synthesis was greater (P < 0.05- < 0.01) in whole blood (leucine data: +50.4 +/- 7.8 nmol/min x 100 ml of forearm; phenylalanine data: +30.4 +/- 11.6) than in plasma (leucine data: +17.8 +/- 5.6 nmol/min x 100 ml of forearm; phenylalanine data: +5.7 +/- 2.1). Consequently, the increment of net amino acid balance was approximately two to fourfold greater (P < 0.04- < 0.03) in whole blood than in plasma. In conclusion, meal ingestion stimulates forearm protein deposition through both enhanced protein synthesis and inhibited proteolysis. Plasma data underestimate net postprandial forearm protein synthesis, suggesting a key role of red blood cells and/or of blood mass in mediating mealenhanced protein accretion.

O.47 Mechanisms of post-prandial protein accretion inhuman skeletal muscle: insight from leucine and phenylalanine forearm kinetics

The relative role of protein synthesis and degradation in de- termining postprandial net protein deposition in human muscle is not known. To this aim, we studied forearm leu- cine and phenylalanine turnover by combining the arterio- venous catheterization with tracer infusions, before and following a 4 h administration of a mixed meal in normal volunteers. Forearm amino acid kinetics were assessed in both whole blood and plasma. Fasting forearm protein deg- radation exceeded synthesis ( P , 0.01) using either tracer, indicating net muscle protein loss. The net negative forearm protein balance was quantitatively similar in whole blood and in plasma. After the meal, forearm proteolysis was sup- pressed ( P , 0.05- , 0.03), while forearm protein synthesis was stimulated ( P , 0.05- , 0.01). However, stimulation of protein synthesis was greater ( P , 0.05- , 0.01) in whole blood (leucine data: 1 50.4 6 7.8 nmol/min 3 100 ml of fore- arm; phenylalanine data: 1 30.4 6 11.6) than in plasma (leu- cine data: 1 17.8 6 5.6 nmol/min 3 100 ml of forearm; phe- nylalanine data: 1 5.7 6 2.1). Consequently, the increment of net amino acid balance was approximately two to fourfold greater ( P , 0.04- , 0.03) in whole blood than in plasma. In conclusion, meal ingestion stimulates forearm protein depo- sition through both enhanced protein synthesis and inhib- ited proteolysis. Plasma data underestimate net postpran- dial forearm protein synthesis, suggesting a key role of red blood cells and/or of blood mass in mediating meal- enhanced protein accretion. ( J. Clin. Invest. 1996. 98:1361- 1372.) Key words: postprandial stateforearm protein syn- thesisforearm proteolysisleucine oxidation

In vivo assessment of mutations in the phenylalanine hydroxylase gene by phenylalanine loading: Characterization of seven common mutations

Mutations in the gene encoding phenylalanine hydroxylase (PAH) cause persistent hyperphenylalaninaemia. To date, more than 200 point mutations and microdeletions have been characterized. Each mutation has a particular quantitative effect on enzyme activity and recessive expression of different mutant alleles results in a marked interindividual heterogeneity of metabolic and clinical phenotypes. In this paper we demonstrate how a simple clinical test can be used to evaluate the correlation between mutation genotype and phenylalanine metabolism. In hyperphenylalaninaemic patients with known PAH mutation genotype, we have investigated phenylalanine turnover in vivo by measuring the ability to eliminate a test dose of L-phenylalanine. All patients could be considered functionally hemizygous for one of their mutant alleles by carrying on the other allele a mutation that is known to completely abolish PAH activity and encode a peptide with no immunoreactivity. Seven mutations (R408W, IVS-12nt1, R261Q, G46S, Y414C, A104D, and D415N) were characterized by oral phenylalanine loading, each mutation being represented by at least three patients. The elimination profile determined for a 3-day period provides a measure to compare residual activity of the mutant proteins and to assign each mutation to a particular metabolic phenotype. The established relation between genotype and phenotype may enable prediction of the severity of the disease by genotype determination in the newborn period. This will aid in the management of hyperphenylalaninaemia and may improve prognosis. The possibility of predicting the residual enzyme activity by DNA analysis performed already in the newborn period allows the prompt implementation of a diet that is adjusted to the degree of PAH deficiency. This may improve management and prognosis of hyperphenylalaninaemia.

Do nonesterified fatty acids regulate skeletal muscle protein turnover in humans?

We examined whether elevated plasma nonesterified fatty acid (NEFA) levels exert a direct effect on protein metabolism by measuring [2H5]phenylalanine skeletal muscle exchange and whole body turnover. [2H5]phenylalanine was infused (0.5 mg.kg-1 x h-1) for 300 min in seven healthy subjects on two occasions. Intralipid (10%; 30 ml/h) or 0.154 mol/l NaCl was infused in random order from 120 min. Measurements were taken during basal (90-120 min) and infusion (270-300 min) periods. Intralipid infusion increased plasma NEFA levels [1.31 +/- 0.13 vs. 0.49 +/- 0.05 (SE) mmol/l; P < 0.05] and forearm NEFA uptake [45 +/- 76 vs. -51 +/- 44 nmol . 100 ml forearm-1 x min-1; P < 0.05]. Serum insulin and blood ketone body levels were similar with the two treatments. Elevated plasma NEFA levels were associated with a comparable decrease in forearm phenylalanine uptake (11 +/- 2 vs. 17 +/- 2 nmol x 100 ml forearm-1 x min-1; lipid vs. control, P < 0.05) and release (20 +/- 2 vs. 26 +/- 3 nmol x 100 ml forearm-1 x min-1; lipid vs. control, P < 0.05). However, there were no significant changes in net forearm phenylalanine exchange and whole body phenylalanine turnover. Therefore, elevated plasma NEFA levels were associated with a comparable decrease in the rates of skeletal muscle protein synthesis and breakdown but did not appear to influence overall protein balance, as assessed using [2H5]phenylalanine.

Ecdysteroids affect in vivo protein metabolism of the flight muscle of the tobacco hornworm ( Manduca sexta)

Ecdysteroid growth promotion of the dorsolongitudinal flight muscle of Manduca sexta was studied by measuring in vivo protein metabolism using both “flooding-dose” and “non-carrier” techniques. These procedures differ in that the former method includes injection of non-labelled phenylalanine (30 μmol/insect) together with the [ 3H]amino acid. Injected radioactivity plateaued in the haemolymph within 7 min. With the flooding-dose method, haemolymph and intramuscular specific radioactivities were similar between 15 min and 2 h. Incorporation of [ 3H]phenylalanine into muscle protein was linear with either method between 30 and 120 min. Fractional rates (%/12 h) of synthesis with the flooding-dose technique were best measured after 1 h because of the initial delay in radioactivity equilibration. Estimation of body phenylalanine turnover with the non-carrier method showed 24–53%/h which was negligible with the flooding-dose method. Since the two methods yielded similar rates of protein synthesis, the large injection of non-labelled amino acid did not alter the rate of synthesis. Because the flooding-dose technique requires only a single time point measurement, it is the preferred method. The decline and eventual cessation of flight-muscle growth was mostly a consequence of declining protein synthesis though degradation increased between 76–86 h before eclosion and was relatively rapid. This decline in muscle growth could be prevented by treating pupae with 20-hydroxyecdysone (10 μg/insect). Protein accretion was promoted by a decline of up to 80% in protein breakdown, which was offset in part by a concurrent though much smaller decrease in protein synthesis. Therefore, ecdysteroids may increase flight-muscle growth by inhibiting proteolysis.

Metabolic regulation of amino acid uptake in marine waters1

To determine the relationships among the processes of uptake, intracellular pool formation, and incorporation of amino acids into protein, we measured the uptake of dipeptides and free amino acids by bacterial assemblages in estuarine and coastal waters of the southeast U.S. The dipeptide phenylalanyl‐phenylalanine (phe‐phe) lowerd Vmax of phenylalanine uptake when the turnover rate of phenylalanine was relatively high. When the turnover rate was relatively low, phe‐phe either had no effect or increased Vmax of phenylalanine uptake. An analytical model was developed and tested to measure the turnover time of the intracellular pool of phenylalanine. When phe‐phe was not added the turnover time of the intracellular pool was 0.89 min. Addition of 1.0 nM phe‐phe increased the turnover time of this pool to 3.8 min in waters where phe‐phe lowered Vmax of phenylalanine uptake. These results suggested that the size of the intracellular pool is regulated, which precludes high assimilation rates of both phenylalanine and phe‐phe. In waters with relatively low phenylalanine turnover rates, bacterial assemblages appear to have a greater capacity to assimilate phenylalanine and phe‐phe simultaneously. Marine bacterial assemblages do not substantially increase the apparent respiration of amino acids when concentrations increase. We conclude that sustained increases in uptake rates and mineralization by marine bacterial assemblages in response to an increase in the concentration of dissolved organic nitrogen is determined by the rate of protein synthesis.

Formation of hippuric acid from phenylalanine labelled with carbon-14 in phenylketonuric subjects.

VARIOUS authors have recently demonstrated the formation of hippuric acid from endogenous benzoic acid in the mammalian organism. Fasting humans1 as well as humans and rats on a lactalbumin–carbohydrate diet2 excreted hippuric acid, and its formation in the intact rat could not be inhibited by sulphasuxidine2 or chloramphenicol3. Application of deuterium and phenylalanine labelled with carbon-14 resulted in labelled urinary hippuric acid2–4. In addition, when benzoic acid was isolated from the urine of human or rats fed benzoic acid, labelled with deuterium or carbon-14, the specific activity was about 20 per cent lower, indicating dilution by endogenous benzoic acid4,5. During the investigation of phenylalanine turnover in human phenylketonuria6, I had the opportunity of studying the formation of hippuric acid from phenylalanine. Phenylketonuric subjects are characterized by their inability to convert phenylalanine to tyrosine to any significant extent. This results in an accumulation of phenylalanine and its keto derivative, phenylpyruvic acid.