Clinical usefulness of urinary 3-methylhistidine excretion in indicating muscle protein breakdown.

Urinary excretion of the post-translationally modified amino-acid 3-methylhistidine, derived from the contractile proteins actin and myosin, was measured in patients with conditions associated with nitrogen loss. The ratio of 3-methylhistidine:creatinine excretion, a measure of the fractional catabolic rate of myofibrillar protein was increased in severe injury, thyrotoxicosis, neoplastic disease, prednisolone administration, and sometimes Duchenne muscular dystrophy. In myxoedema, osteomalacia, and hypothermia the ratio was decreased; and starvation, elective operations, and rheumatoid arthritis had little effect. Provided that the diet is meat free, measurement of urinary 3-methylhistidine may provide useful information on the cause of protein loss.     

Involvement of the liver in the regulation of tryptophan availability. Possible role in the responses of liver and brain to starvation

The concentrations of free and total (free plus albumin bound) tryptophan were measured in plasma of blood taken from the portal vein, hepatic vein and abdominal aorta of male rats, fed, and starved for one and three days. Liver and brain tryptophan concentrations were measured in similar groups of rats.On starvation, there was an increase in arterial plasma free tryptophan concentration which took place peripherally and was paralleled by an increase in brain tryptophan. In both the fed and starved rats, the portal vein concentrations of free tryptophan were high and as the blood flowed through the liver they were reduced to relatively low levels not directly related to the arterial values. All these changes were due to alterations in degree of binding of tryptophan to plasma albumin.The measurements of plasma total tryptophan concentrations showed that postabsorptively and during starvation there was a net uptake of tryptophan by the peripheral tissues (which included brain), but no overall fall in plasma concentration. At the same time, there was a net release from the liver, and to a lesser extent from the portal-drained tissues. The released tryptophan largely entered the albumin bound plasma pool. Accompanying the hepatic output was a fall in tryptophan concentration in the liver which was apparently caused by altered cell membrane transport.The results suggest (1) that the liver protects the brain from the high free tryptophan level in portal blood, (2) that the availability of tryptophan to the brain is maintained postabsorptively and during starvation by hepatic output into the albumin bound pool and (3) that this release of tryptophan from the liver and the fall in intracellular tryptophan concentration are initiated by altered membrane transport. The pattern of changes is consistent with a role for tryptophan in the mediation of changes in liver protein synthesis and gluconeogenesis and cerebral serotonin turnover on starvation.     

Pyridoxine supplementation corrects vitamin B6 deficiency but does not improve inflammation in patients with rheumatoid arthritis

Patients with rheumatoid arthritis have subnormal vitamin B6 status, both quantitatively and functionally. Abnormal vitamin B6 status in rheumatoid arthritis has been associated with spontaneous tumor necrosis factor (TNF)-α production and markers of inflammation, including C-reactive protein and erythrocyte sedimentation rate. Impaired vitamin B6 status could be a result of inflammation, and these patients may have higher demand for vitamin B6. The aim of this study was to determine if daily supplementation with 50 mg of pyridoxine for 30 days can correct the static and/or the functional abnormalities of vitamin B6 status seen in patients with rheumatoid arthritis, and further investigate if pyridoxine supplementation has any effects on the pro-inflammatory cytokine TNF-α or IL-6 production of arthritis. This was a double-blinded, placebo-controlled study involving patients with rheumatoid arthritis with plasma pyridoxal 5'-phosphate below the 25th percentile of the Framingham Heart Cohort Study. Vitamin B6 status was assessed via plasma and erythrocyte pyridoxal 5'-phosphate concentrations, the erythrocyte aspartate aminotransferase activity coefficient (αEAST), net homocysteine increase in response to a methionine load test (ΔtHcy), and 24 h urinary xanthurenic acid (XA) excretion in response to a tryptophan load test. Urinary 4-pyridoxic acid (4-PA) was measured to examine the impact of pyridoxine treatment on vitamin B6 excretion in these patients. Pro-inflammatory cytokine (TNF-α and IL-6) production, C-reactive protein levels and the erythrocyte sedimentation rate before and after supplementation were also examined. Pyridoxine supplementation significantly improved plasma and erythrocyte pyridoxal 5'-phosphate concentrations, erythrocyte αEAST, urinary 4-PA, and XA excretion. These improvements were apparent regardless of baseline B6 levels. Pyridoxine supplementation also showed a trend (p < 0.09) towards a reduction in post-methionine load ΔtHcy. Supplementation did not affect pro-inflammatory cytokine production. Although pyridoxine supplementation did not suppress pro-inflammatory cytokine production in patients with rheumatoid arthritis, the suboptimal vitamin B6 status seen in rheumatoid arthritis can be corrected by 50 mg pyridoxine supplementation for 30 days. Data from the present study suggest that patients with rheumatoid arthritis may have higher requirements for vitamin B6 than those in a normal healthy population.     

Red cell ferritin content: a re-evaluation of indices for iron deficiency in the anaemia of rheumatoid arthritis.

In iron deficiency anaemia basic red cell content of ferritin is appreciably reduced. This variable was determined in 62 patients with rheumatoid arthritis to evaluate conventional laboratory indices for iron deficiency in the anaemia of rheumatoid arthritis. For 23 patients with rheumatoid arthritis and normocytic anaemia irrespective of plasma ferritin concentration, red cell ferritin content did not differ significantly from that for non-anaemic patients with rheumatoid arthritis. For 27 patients with rheumatoid arthritis and microcytic anaemia, the mean red cell ferritin content for patients with a plasma ferritin concentration in the 13-110 micrograms/l range was appreciably reduced. It was indistinguishable from that for patients with rheumatoid arthritis and classical iron deficiency anaemia, indicated by plasma ferritin concentrations of less than 12 micrograms/l. In contrast, the mean red cell ferritin content for patients with rheumatoid arthritis, microcytic anaemia, and plasma ferritin concentrations above 110 micrograms/l did not differ from that for patients with rheumatoid arthritis and normocytic anaemia. Oral treatment with iron in patients with rheumatoid arthritis, microcytic anaemia, and appreciably reduced red cell ferritin concentrations was accompanied by significant increases in haemoglobin concentration (p less than 0.01), mean corpuscular volume (p less than 0.01), and red cell ferritin contents (p less than 0.05). This treatment, however, did not produce any appreciable change in haemoglobin concentration in patients with rheumatoid arthritis, normocytic anaemia, and normal red cell ferritin contents. These findings suggest that the indices for iron deficiency in patients with rheumatoid arthritis and anaemia should include peripheral blood microcytosis together with a plasma ferritin concentration of less than 110 micrograms/l.     

Amino acid composition of human liver mitochondrial membranes in normal and pathological conditions

The amino acid composition of proteins from liver mitochondrial membranes has been studied in patients with normal liver, with biliary diseases and fatty liver, with obstructive jaundice or liver cirrhosis. A characteristic pattern of the amino acid composition in patients with normal liver has been found. In the mitochondrial membranes of patients with fatty liver tryptophan and lysine were decreased while [aspartic acid plus asparagine] and [glutamic acid plus glutamine] were increased compared to their counterpart in the normal liver. In patients with obstructive jaundice of short duration (less than two months) only a slight decrease in methionine content was found, while in the case of liver cirrhosis amino acid composition was markedly changed.deceased.     

Effect of Chronic Corn Consumption on Serotonin Content of Rat Brain

SEROTONIN, a putative neurotransmitter in the mammalian central nervous system, is synthesized in the brain by the 5-hydroxylation and decarboxylation of the essential amino-acid L-tryptophan^1,2. The control of serotonin biosynthesis seems to involve a different mechanism from that responsible for catecholamine biosynthesis^3,4 in its dependence on the availability of the amino-acid precursor^5,6. Thus, small doses of tryptophan that do not increase brain or plasma tryptophan concentrations beyond their normal daily ranges cause significant increases in the serotonin concentration of rat brain⁷. Conversely, the chronic ingestion of diets lacking in tryptophan (with casein hydrolysates or amino-acid mixtures substituted for natural proteins) depresses brain serotonin levels^8–10. The dependence of serotonin biosynthesis on tryptophan availability probably arises from the unusually high substrate K _M that characterizes tryptophan hydroxylase¹. It seems likely that this enzyme normally functions in an unsaturated state; hence physiological increases in intraneuronal tryptophan could drive the hydroxylation of the amino-acid and, ultimately, its conversion to serotonin.     

Rheumatoid Arthritis and Personality: A Controlled Study

Evidence in support of claims for the existence of a special relationship between personality and rheumatoid arthritis is conflicting. In this study four groups—one of patients with early rheumatoid arthritis, one of patients with chronic rheumatoid arthritis, one of neurotic patients, and a normal control group—were compared by means of the Maudsley Personality Inventory (M.P.I.) and a neurotic trait in childhood (N.T.C.) score. Both arthritis groups had a lower M.P.I. neuroticism score than the normal control group, with greater significance in the chronic arthritis group. The neurotic group had a significantly higher neuroticism score than the other three groups. Both arthritis groups had a lower extraversion score than normal controls, again with greater significance in the chronic arthritis group. The neurotic group scored significantly less than normal controls on the extraversion scale and intermediately between the early and chronic arthritis groups. There was no significant difference between the arthritis groups and the normal control group in the N.T.C. score, but it was significantly increased in the neurotic group.These findings suggest that people with rheumatoid arthritis differ significantly in personality from normal and from neurotic people, that the differences are accentuated with chronicity in the rheumatoid process, and that the differences develop as a result of the arthritis.     

Correlation between tryptophan, NEFA and albumins in the nephrotic syndrome

Many connections were considered between bound and free tryptophan and albumins, NEFA and other aminoacids in 18 proofs in ten subjects of paediatric age affected by nephrotic syndrome. A decrease of total tryptophan and a tendency to increase of free tryptophan were showed in our experience. NEFA, at normal concentrations, should not be responsible for this; and this could suggest that the binding sites on albumins for NEFA and tryptophan are different. Besides there is appearance of a positive correlation between albumins and bound tryptophan and a negative correlation between albumins and free tryptophan. These results can suggest that the reduction of the total tryptophan is due to the loss of the fractions bound to albumins, but urinary tryptophan is not increased in our studies. As the albumins get fewer, there is a lost in linked tryptophan and an increase of free tryptophan. A total reduction of other aromatic aminoacids can also explain, through a reduced intestinal absorption, the decrease of the tryptophan in the nephrotic syndrome.     

Nyctotheroides puytoraci: stimulation of encystment in toads, Bufo regularis, injected with rheumatoid arthritis patients' urine.

Nyctotheroides puytoraci, a ciliated protozoan parasite first described by Essawy (M.Sc. thesis, Alexandria University, Alexandria, Egypt, 1978), reacted by encystation in toad hosts Bufo regularis that had been injected subcutaneously with urine of patients with rheumatoid arthritis. It is speculated that carcinogenic tryptophan metabolites present in the injected urine reach parasites in the recta of treated host animals and are the inducers of encystment. Increased encystment was obtained when hosts were injected with the urine of rheumatoid arthritis patients who had been given 2 g l-tryptophan orally. On the other hand, injection of B. regularis with the urine of rheumatoid arthritis patients who had been given 100 mg of pyridoxine HCl did not induce increased cyst formation in the parasite. The abnormality of tryptophan metabolism in rheumatoid arthritis patients was readily corrected by the administration of pyridoxine.     

PLASMA TRYPTOPHAN AND 5-HT METABOLISM IN THE CNS OF THE NEWBORN RAT

—The relationships between plasma tryptophan and 5-HT metabolism in the CNS were studied in newborn rats and compared with adults. Both the concentration of free tryptophan in plasma and that of the amino-acid in brain were much higher immediately after birth than later on. Drugs such as salicylate and chlordiazepoxide, which increased brain tryptophan concentrations in adults by displacing the plasma amino acid bound to serum albumin, were ineffective in newborn rats: most of the amino acid being already free in their plasma. The study of 5-HT metabolism in brain stem slices revealed that the affinity of the uptake process for tryptophan was higher in newborn than in adult animals, whereas the reverse situation was observed for the enzyme complex involved in 5-HT synthesis (lower apparent K_m in adults). In addition, the catabolism of newly synthesized 5-HT was more rapid in newborn than in adult tissues. Finally, the free state of tryptophan in plasma of newborn animals induced in brain both a high amino acid concentration and, in contrast to the situation observed in adults, a synthesis rate of 5-HT very near its maximal value.     

Evidence for defect of complement-mediated phagocytosis by monocytes from patients with rheumatoid arthritis and cutaneous vasculitis.

In-vitro measurements of the rate of monocyte phagocytosis of heat-killed yeast preopsonised in human AB serum from 14 patients with rheumatoid arthritis and 14 normal controls showed a significant reduction in five patients with active vasculitis but no change in nine with active arthritis alone. Further studies of complement- and Fc-mediated monocyte phagocytosis in which the rate constants (Kc and KFc respectively) were determined using complement-coated Saccharomyces cerevisiae and Candida albicans opsonised with IgG in monocytes from nine patients with rheumatoid vasculitis and 12 controls showed a significant reduction in Kc (p less than 0.01) but normal KFc. Kc was normal in three patients with inactive vasculitis. Low Kc was correlated with low serum C3 concentrations but not with Clq binding or anticomplementary activity, and no evidence of intracytoplasmic or membrane-bound immune complexes was detected in monocytes from patients with active vasculitis. These results show that cutaneous vasculitis in rheumatoid arthritis is associated with selective impairment of complement-mediated monocyte phagocytosis, which does not appear to result from receptor blockade by immune complexes.     

The subclass distribution of human IgG rheumatoid factor

The subclass distribution of IgG rheumatoid factor (RF) was determined by a sensitive ELISA assay in sera from patients with rheumatoid arthritis and from normal controls. In both instances, the most important subclasses were IgG1 and IgG4. The IgG4 RF was directed against the Fc region of IgG, and recognized human as well as rabbit IgG. Although human IgG4 myeloma proteins bound to rabbit IgG better than did myelomas of other IgG subclasses, the IgG4 RF activity in rheumatoid sera showed an additional specificity, because the fraction of IgG4 RF/total IgG4 for rheumatoid arthritis sera was far greater than for myelomas. This inference was supported by the observation that there was persistent, albeit diminished, IgG RF activity in pepsin-digested, RF-containing sera (but not myeloma proteins), indicating that a critical component of IgG4 RF activity was contained within the Fab region of the IgG4 molecule. The finding of large quantities of IgG4 RF was not due to a bias of the assay, because the preponderance of IgG4 did not extend to the subclass distribution of antibodies directed against other antigens. The demonstration of an important role for IgG4 as a RF is of special interest because of the relative inability of this subclass to fix complement or to bind to Fc receptors, and because of its potential role as a mediator of increased vascular permeability.     

Oxygen radical induced fluorescence in proteins; identification of the fluorescent tryptophan metabolite,N-formyl kynurenine,as a biological index of radical damage

Summary The effect of oxygen derived free radicals (OFR) on aromatic and sulphur containing amino acids has been investigated, both in their free form and within protein backbones. Aerated amino acids and proteins in solution were exposed to three discrete OFR generating systems; (1) gamma radiation in the presence or absence of formate (2) photolysis by UV light at 254 and 366 nm, and (3) site specific modification by H₂O₂ in the presence of CuII ions.A sensitive reverse-phase HPLC technique with dual detection systems (UV absorbance and fluorescence monitoring) was developed to analyse the products of amino acid oxidation. OFR denatured amino acids were chromatographed by this procedure, and all radical species generated, with the exception of the superoxide anion, resulted in the formation of identifiable fluorescent metabolites of tryptophan, kynurenines. The identity of peaks was confimed by spiking with authentic material and scanning absorption spectroscopy. After complete proteolytic hydrolysis, OFR treated proteins were also analysed by this technique; again the dose dependent production of kynurenines was detected in IgG, lens crystallins and albumin. Bityrosine was not detected in any of the proteins studied using this procedure, however, several novel unidentified fluorophores were detected in proteolytic hydrolysates, possibly the product of two different amino acid radicals.Immunoglobulin G isolated from the sera of normals and rheumatoid arthritis (RA) patients was examined for the presence of one specific tryptophan metabolite, N-formyl kynurenine. Significantly elevated levels of this metabolite were detected in rheumatoid sera, suggesting increased OFR activity in RA.These results have demonstrated firstly, that specific oxidised products of amino acids are retained in the protein backbone after exposure to OFR generating systems. Secondly, in aerated solution, oxidised tryptophan residues confer the major new visible fluorescence in non-haem proteins, not tyrosine products. In addition, this work has demonstrated that the measurement of a specific product of an oxidised amino acid can be applied to biological macromolecules, and may be important in implicating free radical reactions in certain disease processes.     

Inhibition of indoleamine 2,3-dioxygenase-mediated tryptophan catabolism accelerates collagen-induced arthritis in mice

Indoleamine 2,3-dioxygenase (IDO) is one of the initial and rate-limiting enzymes involved in the catabolism of the essential amino acid tryptophan. In cultured cells, the induction of IDO leads to depletion of tryptophan and tryptophan starvation. Recent studies suggest that modulation of tryptophan concentration via IDO plays a fundamental role in innate immune responses. Induction of IDO by interferon-γ in macrophages and dendritic cells results in tryptophan depletion and suppresses the immune-mediated activation of fibroblasts and T, B, and natural killer cells. To assess the role of IDO in collagen-induced arthritis (CIA), a model of rheumatoid arthritis characterized by a primarily Th1-like immune response, activity of IDO was inhibited by 1-methyl-tryptophan (1-MT) in vivo. The results showed significantly increased incidence and severity of CIA in mice treated with 1-MT. Activity of IDO, as determined by measuring the levels of kynurenine/tryptophan ratio in the sera, was increased in the acute phase of arthritis and was higher in collagen-immunized mice that did not develop arthritis. Treatment with 1-MT resulted in an enhanced cellular and humoral immune response and a more dominant polarization to Th1 in mice with arthritis compared with vehicle-treated arthritic mice. The results demonstrated that development of CIA was associated with increased IDO activity and enhanced tryptophan catabolism in mice. Blocking IDO with 1-MT aggravated the severity of arthritis and enhanced the immune responses. These findings suggest that IDO may play an important and novel role in the negative feedback of CIA and possibly in the pathogenesis of rheumatoid arthritis.     

Possible relationship between pH of plasma and rheumatoid arthritis.

The free level of tryptophan in untreated Rheumatoid Arthritic patients is lower than normal controls and normal subjects have a daily fluctuation in the concentration of the amino-acid. The present study shows that both of these changes could be caused by small changes in the pH of human plasma which fall within the physiological range of pH found in blood.     

Reduced amyloid-A-degrading activity in serum in amyloidosis associated with rheumatoid arthritis.

The ability to degrade amyloid A fibrils was studied in the serum of 31 patients with amyloidosis associated with rheumatoid arthritis, 33 patients with rheumatoid arthritis without amyloidosis, and 47 healthy controls. Fibrillar amyloid A protein and the radial diffusion method were used. The mean degrading activity in serum was significantly lower in patients with rheumatoid arthritis complicated by amyloidosis (58 +/- 19% SD of the activity in a pooled sample of sera from 100 healthy blood donors used as standard) than in patients with rheumatoid arthritis alone (78 +/- 14%; p less than 0.001) or controls (99 +/- 19%; p less than 0.001). Alpha 1-antitrypsin, concentrations of which were raised in both groups of patients, inhibited the degrading activity in serum even in low concentrations. A negative correlation between degrading activity and alpha 1-antitrypsin concentrations was observed. These findings suggest that reduced amyloid-A-degrading activity is due to inhibition rather than to deficiency of enzyme.     

Differential synthesis of 5-lipoxygenase in peripheral blood and synovial fluid neutrophils in rheumatoid arthritis

In order to investigate 5-lipoxygenase enzyme regulation in neutrophils during an inflammatory reaction, we studied 5-lipoxygenase mRNA levels, as well as de novo enzyme synthesis, in resting and activated neutrophils isolated from normal individuals and patients with rheumatoid arthritis. The approach used was to analyze these activities in resting peripheral blood neutrophils of normal individuals on the one hand and in peripheral blood and matched synovial fluid neutrophils isolated from patients with rheumatoid arthritis on the other hand. Our first observation was that resting peripheral blood neutrophils of either normal individuals or patients show detectable levels of 5-lipoxygenase mRNA and are able to synthesize the enzyme de novo. Our second observation was that inflammatory activated neutrophils from synovial fluid reveal lower 5-lipoxygenase mRNA levels and enzyme synthesis than do the patient-matched peripheral blood cells. This is in spite of the fact that, for other proteins, synovial fluid neutrophils are equally or more active than their peripheral blood counterparts. We conclude that peripheral blood neutrophils are capable of synthesizing the enzyme, thus ensuring the turnover of the protein. Furthermore, complex regulatory mechanisms appear to take place in response to inflammation as it occurs in synovial fluids of patients with rheumatoid arthritis, leading to decreased mRNA levels and enzyme synthesis. Possible mechanisms of regulation are discussed and are presently under investigation.     

A pattern of protein expression in peripheral blood mononuclear cells distinguishes rheumatoid arthritis patients from healthy individuals

We compared the expression levels of proteins in peripheral blood mononuclear cells (PBMCs) of healthy control individuals to those of patients diagnosed with rheumatoid arthritis (RA) using a proteomics approach. Using two-dimensional electrophoresis we identified 18 proteins that were 2-fold or more highly expressed in patients than in controls, and 11 proteins that were 2-fold or more highly expressed in controls than in patients. Some of these differentially expressed proteins, identified by MALDI-TOF spectrometry, have previously been shown to play a potential role in the pathogenesis of RA. Hierarchical cluster analyses of the data segregated the samples into two groups, one which contained only controls and the other which contained only patients, and was used to compare the expression pattern of these 29 proteins in individual samples with the median expression pattern determined in the healthy control and in the RA patient groups. This analyses was able to predict whether a sample was derived from a rheumatoid arthritis patient or from a healthy individual, suggesting that a comparison of such protein expression patterns may be of diagnostic value.     

Effects of Albumin, Amino Acids and Clofibrate on the Uptake of Tryptophan by the Rat Brain

Abstract: The influences of total tryptophan concentration, albumin binding and amino acid competition on the rate of tryptophan influx into rat brain were compared using a single-pass injection technique with tritiated water as a freely diffusible reference. Omission of 3% bovine albumin from a bolus containing tryptophan in Krebs–Ringer bicarbonate buffer injected into the carotid artery increased non-albumin bound (free) tryptophan concentration threefold but tryptophan uptake by only 35% and 30% into forebrain and hypothalamus, respectively. However, tryptophan uptake from injected rat plasma was more markedly elevated when free tryptophan concentration was raised. Thus, when free tryptophan was doubled, but total tryptophan unchanged, by in vitro addition of clofibrate to a plasma bolus, uptake was increased by 53% and 28% into forebrain and hypothalamus respectively. When clofibrate was injected in vivo so that plasma total tryptophan concentration was decreased by 45% but neither free tryptophan nor competing amino acid concentrations were altered, then uptake from a bolus of the rat's own plasma was unchanged. Addition of competing amino acids at physiological concentrations to tryptophan in Krebs-Ringer buffer significantly reduced tryptophan influx into both brain regions, but did not increase the effect of albumin binding. The results indicate that tryptophan uptake into rat forebrain is substantially influenced by albumin binding and competition from other amino acids, but that hypothalamic uptake is less influenced by these factors.     

Impact of potassium,sodium, and salinity on the protein-and free amino acid content of wheat grain

A lysimeter study was conducted on Cajeme wheat (Triticum aestivum L.) to investigate the impact of salinity on protein and free amino acid content of the grain. Cross correlations were obtained between 16 different soil-plant-water based parameters and the concentration and total accumulation of amino acids. The results indicated that after 3 years of irrigation, the majority of protein bound and free amino acids increased in concentration in the grain. However, both free tryptophan and free proline revealed decreasing concentrations with increasing salinity. Free tryptophan showed a synergism between total accumulation, yield and concentration. Free proline concentrations decreased in association with increasing protein concentrations. Cross correlations of the 16 soil-plant-water based parameters with free and protein bound amino acids revealed significant correlations for free aspartic acid and glycine with total accumulation but not with concentrations. Only methionine plus cystine was lower than suggested FAO levels for essential amino acids and was lower in the third year than in the first year.