The need of essential amino acids in children. An evaluation based on the intake of phenylalanine, tyrosine, leucine, isoleucine, and valine in children with phenylketonuria, tyrosine amino transferase defect, and maple syrup urine disease.

The diet of children with blocks in the metabolism of five amino acids has been investigated to evaluate the need for these amino acids in the maintenance of normal growth and development. Two children with phenylketonuria, one child with tyrosine aminotransferase defect and one child with maple syrup urine disease are included in the study. The growth and development of the children have been within the normal range except for language development, which was retarded in the maple syrup urine disease child. The need for phenylalanine, phenylalanine and tyrosine combined and isoleucine, leucine, and valine for protein synthesis in growing children was investigated by registering the intake of phenylalanine in the phenylketonuria children, the intake of phenylalanine and tyrosine in the tyrosine amino transferase defect child and isoleucine, leucine and valine in the maple syrup urine disease child. The significance of this intake, defined as the sufficient intake, is discussed, as well as the difference between the sufficient intake and requirement. The sufficient intake is compared with former studies on requirement. There is good agreement between the sufficient intake and requirement of phenylalanine and tyrosine. The sufficient intake of isoleucine, leucine, and valine as judged from our study is lower than in former studies on requirement.

Reduced nutritional status in an elderly population (> 70 y) is probable before disease and possibly contributes to the development of disease.

In this prospective study of elderly people, nutritional status (body mass index, triceps skinfold thickness, arm-muscle circumference, and serum albumin) was assessed in a group of recently hospitalized (< 48 h) patients (n = 311), and compared with a home-living group (n = 106). Undernutrition was present in 52.9% of males and 60.6% of females by the time of admission to the hospital. Further, 65% of the males and 69% of the females had an insufficient energy intake the month before hospitalization [males < 8372 kJ (2000 kcal) and females < 7116 kJ (1700 kcal)]. Intake of vitamins and trace elements below two-thirds of the US recommended dietary allowances was more common in the hospital group. This group was more often unable to buy food and cook dinner, had more chewing problems, and had reduced appetite for food. Reduced nutrient and energy intakes may increase the occurrence of undernutrition, with increased risk for hospitalization in vulnerable groups as a consequence.

Protein requirements in infants and children: a longitudinal study of children treated for phenylketonuria.

Two groups of children with phenylketonuria were followed from birth for several years. The Recommended Dietary Allowance group received a protein intake as recommended by the Food and Nutrition Board. The Food and Agricultural Organization (FAO) group received a protein intake as recommended by FAO. The children were followed very closely for the biochemical control of the disease. The children were also followed very closely to evaluate the adequacy of the protein intake using length, weight, routine hematology, chemical analysis, and x-ray of the hand. The results indicated two groups of healthy children. However, a decline in length growth percentile was found in some of the FAO children. A possible osteoporosis developed in two of the FAO children. The possible conclusion that the FAO "safe level of intakes of egg or milk protein" is marginal is discussed.

Molecular sieving and mass spectroscopy reveal enhanced collagen degradation in rabbit atheroma.

BACKGROUND: Collagen degradation is the major mechanism of atherosclerotic plaque destabilization. It is unknown whether collagen breakdown is involved into formation of early atherosclerotic lesions. METHODS: Current paper describes a novel collagen degradation assay based on a combination of molecular sieving and mass spectroscopy. The first step of the assay consists of the extraction of total collagen from tissue. This extract includes both intact collagen and its breakdown products. Molecular sieving is used to isolate low molecular weight collagen fragments. Since the low molecular weight fraction of the extract may contain some non-collagenous molecular species, the collagen-specific amino acid hydroxyproline is quantified using mass spectroscopy. RESULTS: This assay was validated in various experimental systems with known/predictable level of collagen breakdown in vitro, ex vivo and in vivo. When applied to cholesterol-fed rabbit aorta, it revealed enhanced collagen degradation in rabbit atheromas compared to unaffected aortic regions. CONCLUSION: A novel assay has been developed to demonstrate enhanced collagen degradation in rabbit atherosclerotic plaques. Accurate quantification of collagen breakdown products should provide a new relevant end point in the analysis of plaque development and stability.

Troglitazone quinone formation catalyzed by human and rat CYP3A: an atypical CYP oxidation reaction.

Oxidative ring opening of troglitazone (TGZ)(1) a thiazolidine 2,4-dione derivative used for the treatment of type II diabetes mellitus, leads to the formation of a quinone metabolite. The formation of TGZ quinone was shown to be NADPH dependent and to require active microsomal enzymes. Quinone formation was not affected by co-incubation with catalase or sodium azide and was partially inhibited (25%) by superoxide dismutase (SOD). Kinetic analysis of TGZ quinone formation in human liver microsomes implied single enzyme involvement. CYP3A isoforms were characterized as the primary enzymes involved in quinone formation by several lines of evidence including: (a) troleandomycin and ketoconazole almost completely inhibited microsomal quinone formation when SOD was present, whereas other CYP inhibitors had minimal effects (<20%); (b) TGZ quinone formation was highly correlated with regard to both contents (r(2): 0.9374) and activities (r(2): 0.7951) of CYP3A4 in human liver microsomes (HLM); (c) baculovirus insect cell-expressed human CYP3A4 was able to catalyze TGZ quinone formation at a higher capacity (V(max)/K(m)) than other human CYPs with the relative contribution of CYP3A4 in HLM estimated to be 20-fold higher than that of other CYPs; (d) TGZ quinone formation was increased by 350% in liver microsomes from rats pretreated with dexamethasone (DEX); and (e) plasma concentrations of TGZ quinone were increased by 260-680% in rats pretreated with DEX. The chemical nature of the quinone metabolite suggests an atypical CYP reaction consistent with a one-electron oxidation mechanism where an intermediate phenoxy radical combines with ferryl oxygen to subsequently form the quinone metabolite.

An improved sensitive assay for simultaneous determination of plasma haloperidol and reduced haloperidol levels by liquid chromatography using a coulometric detector.

A simple, highly sensitive and specific high-performance liquid chromatographic method that uses a coulometric detector for the simultaneous assay of haloperidol and reduced haloperidol in human plasma has been developed, using bromoperidol as the internal standard. A reversed phase C8 5-microns column (25 x 0.46 cm) and a mobile phase of phosphate buffer (pH 7.0), acetonitrile, and methanol (40:20:40 vol/vol) are used for separating the analytes. The analytes are extracted from alkalinized plasma using a mixture of pentane and isopropanol (95:5 vol/vol) and purified by back extraction into a perchloric acid solution. Teflon tubes with screw caps are used throughout the extraction work. The compounds are oxidized at a potential of +0.90 V against a Ag/AgCl reference electrode. The detection limit of the assay is 50 ng/L using 1 mL of plasma. The average interassay coefficient of variation for samples of concentration 1-40 micrograms/L is approximately 8%. Possible drug interferences in the assay have been studied. The absolute recovery of the method is approximately 80%. The assay has been validated by quantitating 150 schizophrenic patients' samples.

Is phenylalanine requirement in infants and children related to protein intake?

Two groups of children with phenylketonuria (PKU) received protein at two different levels. The protein source was a protein hydrolysate, devoid of phenylalanine, and intact protein from milk, vegetables and fruit. One group (RDA group) was given protein at a level based on the recommendations of the (US) Food and Nutrition Board (1974, 1980). The other group (FAO group) was given protein at the level of intake corresponding to the Joint FAO/WHO ad hoc Expert Committee (1973) safe levels of intake of egg or milk protein. The children were monitored very closely for several years. From an earlier study evaluating the protein intake of the two groups it was suspected that the Joint FAO/WHO ad hoc Expert Committee (1973) recommendations were marginal. In the present study the phenylalanine intake of the two groups required to maintain the plasma phenylalanine concentration at the required level was established. The results showed that the RDA group required more phenylalanine than the FAO group. This difference was statistically significant from the age of 5-15 months. We have interpreted the greater requirement for phenylalanine in the RDA group as a result of a greater nitrogen intake and thus a more rapid chemical maturation of N (increase in protein concentration of the body with age). It is known that up to the age of 6 months the chemical maturation of N is related to the N intake. In the present study we have found that this difference in chemical maturation lasted up to the age of 15 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous assay of corticosterone and cortisol in plasma by reversed-phase liquid chromatography.

We have developed a simple, specific, and sensitive reversed-phase liquid-chromatographic method for accurate and simultaneous analysis of corticosterone and cortisol in human plasma. We achieved a detection limit of 300 ng/L for both steroids by modifying the old solid-phase extraction method to make use of "Tef Elutor" C18 columns, using a minibore (100 x 2 mm) analytical column, and using an ultraviolet detector with a 10-mm-pathlength flow cell. With the new extraction method absolute extraction efficiencies were greater than 90% for all the analytes, including the internal standard, flumethasone. The mobile phase was water (containing 5 mL of triethylamine per liter and citric acid to adjust the pH to 6.5), tetrahydrofuran, and acetonitrile (82/10/8 by vol). The average interassay CV for corticosterone at 0-25 micrograms/L was 6.5%; that for cortisol at 0-300 micrograms/L was 3.8%. The analytical recovery relative to the internal standard was 100.2% for cortisol and 102.6% for corticosterone. Possible interferences from drugs and other steroids were studied.

A simple, sensitive liquid chromatographic assay of cis-thiothixene in plasma with coulometric detection.

A novel, simple, and very sensitive liquid-chromatographic assay with a coulometric detector has been developed for quantitating cis-thiothixene (CTX) in human plasma. A reverse phase, 5-microns cyano column (25 x 0.46 cm), a mobile phase of phosphate buffer (pH 2.5) and acetonitrile (40/60 by vol), and a coulometric detector are used for the separation of CTX, and the internal standard, trifluoperazine. CTX and trifluoperazine are extracted from alkalinized plasma into n-pentane-isopropanol (95/5 by vol) and purified by back extraction into perchloric acid. The optimum oxidation potential for the analytes is +0.8 V versus an Ag/AgCl electrode. The detection limit for CTX is 200 pg using 1 mL of plasma and CTX concentrations are linear from 0 to 40 micrograms/L. The average interassay CV is 9%, and the mean recovery is 99% relative to the internal standard. Possible interferences from various psychiatric and common drugs in the assay have been studied. The assay method was validated by determining the concentration of CTX in the plasma of 100 schizophrenic patients.

Net protein utilization determined by rat bioassay of a protein hydrolysate and a diet for children with phenylketonuria.

In a previous study (Kindt et al. 1983, 1984) it was assumed that a protein hydrolysate, devoid of phenylalanine, together with intact protein as given to children with phenylketonuria (PKU), was equivalent to egg or milk protein. One group of children was given this 'PKU protein' in amounts corresponding to the Joint FAO/WHO ad hoc Expert Committee (1973) recommendations. The results indicated that the Joint FAO/WHO ad hoc Expert Committee (1973) recommended levels of protein intake were marginal. The purpose of the present study was to evaluate whether the quality of the protein hydrolysate, together with intact protein ('PKU protein'), is equivalent to egg or milk protein. This was done using a rat bioassay. Four protein sources were used: (1) egg protein, (2) protein hydrolysate, (3) protein hydrolysate diluted with non-essential amino acids, (4) protein hydrolysate mixed with food proteins ('PKU protein'), comparable with the diet previously used (Kindt et al. 1983, 1984). The results indicated that the 'PKU protein' was of very high quality: net protein utilization (NPU) greater than 90. The protein hydrolysate alone and the protein hydrolysate diluted with non-essential amino acids gave a NPU greater than 80. The conclusion drawn from the present study is that the 'PKU protein', as used in the treatment of children with PKU, is equivalent to egg or milk protein. This supported the view that the Joint FAO/WHO ad hoc Expert Committee (1973) recommended levels of intake were marginal.

Assay of human plasma cortisone by liquid chromatography: normal plasma concentrations (between 8 and 10 a.m.) of cortisone and corticosterone.

A high-performance liquid chromatographic method using ultraviolet detection to quantitate human plasma concentrations of cortisone simultaneously with cortisol and corticosterone is described. The method is based on the use of an octadecyl silica column (100 mm x 2 mm I.D., 3 microns), an ultraviolet absorbance detector (242 nm) with a 10 mm path length flow-cell, and a mobile phase composed of water-tetrahydrofuran-acetonitrile (82:10:8, v/v) containing 5 ml/l triethylamine and citric acid to adjust the pH of the buffer to 6.5. Flumethasone is used as the internal standard. The detection limit of the method for the three steroids is 300 ng/l using a 1-ml sample. The average inter-assay coefficient of variation for cortisone is 3.3% and the average recovery is 100.8%. Possible interferences from common drugs and endogenous and exogenous steroids in the method have been studied. Plasma concentrations (drawn from 8 to 10 a.m.) of cortisone and corticosterone for 43 normal volunteers have been determined.

[Children with phenylketonuria (Fölling's disease). Intellectual functions and psychological adaptation]. / Barn med fenylketonuri (Føllings sykdom). Intellektuell funksjon og psykologisk tilpasning.

Untreated phenylketonuria (PKU) leads to serious mental retardation. The prognosis of PKU has been dramatically improved by neonatal screening and dietary treatment. This study evaluates 25 children, ages 10 to 16 years. Children who receive early and adequate treatment have a mean IQ slightly below normal, and few psychological problems. The disease causes considerable strain on the families, however, because of the very strict diet. Recent evidence suggests that children with PKU can only rarely terminate the diet, although in many cases the diet can be relaxed. It is essential to teach the children the diet and promote autonomy in relation to phenylketonuria.

Fasting plasma amino acid concentrations in PKU children on two different levels of protein intake.

Two groups of children with phenylketonuria were treated with different amounts of protein. One group (RDA group) received protein as recommended by the U.S. Food and Nutrition Board, the other (FAO group) the amount recommended by the FAO/WHO 1973 ad hoc Expert Committee. Two of the children in the FAO group showed a decrease in length/growth velocity which raised the question of the adequacy of the protein content in the diet. Since deviations from normal plasma amino acids have been observed in protein deficient, energy-adequate diets, we examined these parameters in the PKU children. In the two children with retardation in length/growth, very high glycine values as well as very high alanine values were observed at the end of the study. These observations strengthened our suspicion that the FAO/WHO 1973 recommendations are marginal.

Quantitative method for biomarkers of collagen degradation using liquid chromatography tandem mass spectrometry.

Preclinical efficacy testing commonly involves studies that require considerable resources and time. One valuable tool in this endeavor is the characterization of relevant biomarkers. A method has been developed for the simultaneous determination of collagen biomarker candidates as an instrument in screening compounds for efficacy. Two potential candidates, the 3-hydroxypyridinium crosslinks pyridinoline and deoxypyridinoline, were selected for analysis in collagen degradation models. Tissue or urine samples were collected, prepared, and quantitated for the biomarkers using spiked calibration curves and liquid chromatography tandem mass spectrometry. The development of a quick and simple assay method would allow us to increase the chances for success in efficacy screening by eliminating compounds with poor biomarker profiles. The method proposed here appears to be more selective, convenient, precise (generally <10% RSD), accurate (generally <10% RE), and sensitive relative to previously established methodology.

The effect of troglitazone biliary excretion on metabolite distribution and cholestasis in transporter-deficient rats.

We investigated whether lack of the canalicular multispecific organic anion transporter in transport-deficient (TR-) rats would result in plasma and urinary accumulation of troglitazone or its major metabolites and whether any accumulation would be associated with increased levels of bilirubin or bile acids. Administration of a single oral dose of troglitazone (200 mg/kg) to TR- rats resulted in 2- and 50-fold increases in plasma levels and 30- and 500-fold increases in urinary amounts of troglitazone sulfate and troglitazone glucuronide, respectively, compared with normal rats. No changes were found in the plasma concentrations and urinary amounts of troglitazone or troglitazone-quinone. Accumulation of troglitazone metabolites in plasma was accompanied by a 2-fold increase in the serum level of conjugated bilirubin in TR- rats, whereas no changes were observed in normal animals. Bile acids were detected in the urine of both TR- and normal rats, with an average 3-fold greater level found in the urine of TR- animals. Biliary metabolic profiles revealed a delay in the secretion of troglitazone sulfate and troglitazone glucuronide in TR- rats over the first 2- and 4-h periods, respectively. These results demonstrate the role of multidrug resistant associated protein-2 in biliary secretion of troglitazone glucuronide and troglitazone sulfate and suggest the presence of compensatory mechanisms responsible for transport of troglitazone metabolites and bilirubin-glucuronide at the basolateral and canalicular sites of hepatocytes.

The role of conjugation in hepatotoxicity of troglitazone in human and porcine hepatocyte cultures.

In primary human and porcine hepatocyte cultures, we investigated the relationship between metabolism and cytotoxicity of troglitazone. Treatment of human hepatocytes for 2 h with 10, 20, 25, 35, and 50 microM troglitazone in protein-free medium resulted in concentration-dependent decreases in total protein synthesis. Decreases at 10 and 20 microM were reversible by 24 h, however protein synthesis did not recover at concentrations >/=25 microM. Troglitazone at 50 microM caused cellular death. In porcine hepatocytes, 100 microM troglitazone was lethal, whereas at 50 microM, protein synthesis completely recovered by 24 h. Recovery in protein synthesis was associated with metabolism of parent drug, whereas toxicity correlated (r(2) = 0.82) with accumulation of unmetabolized troglitazone. By 1 h, in human hepatocytes, troglitazone was metabolized to similar amounts of sulfate and quinone metabolites with little glucuronide detected. In contrast, porcine hepatocytes metabolized troglitazone to the similar amounts of glucuronide and the quinone metabolites with little sulfate detected. Exposure of human hepatocytes to a combination of 10 microM troglitazone and 10 microM 2,4-dichloro-4-nitrophenol resulted in a 70% decrease in protein synthesis, associated with 90% inhibition in the formation of troglitazone sulfate, a 4-fold increase in unmetabolized troglitazone, and no effect on formation of the quinone metabolite. Treatment with a combination of acetaminophen or phenobarbital with 20 microM troglitazone resulted in sustained decrease in protein synthesis associated with inhibition of sulfation and accumulation of troglitazone. These results suggest that inhibition of troglitazone sulfation may result in increased hepatotoxicity due to exposure to parent drug, or increased metabolism by alternate pathways.

Hypercholesterolemia causes mechanical weakening of rabbit atheroma : local collagen loss as a prerequisite of plaque rupture.

Hypercholesterolemia may render atherosclerotic plaques prone to rupture. To test this hypothesis, catheters with matrix-covered balloons were implanted into the aorta of rabbits fed standard or 0. 5% cholesterol chow (n=70). In 1 month, fibrous plaques developed around the balloon. Time-dependent accumulation of cholesteryl esters and free cholesterol was detected in the plaques of the cholesterol-fed group only. The pressure needed to rupture the plaque by balloon inflation was used as an index of plaque strength. Three months after the catheter implantation, the breaking pressure was 2.1 times lower (P<0.05) in cholesterol-fed rabbits. It was accompanied by collagen loss, as measured by plaque hydroxyproline content, but not with deficiency of collagen cross-linking. Sirius red staining showed preservation of collagen originally covering the balloon and accumulation of nascent collagen in the lesions of standard chow-fed rabbits. In the cholesterol-fed group, both mature and new collagen underwent degradation predominantly in the plaque shoulders. Collagen breakdown was associated with local accumulation of foamy macrophages. Gel zymography demonstrated relative enhancement of gelatinolytic activity at 92 and 72 kDa, as well as caseinolytic activity at 57, 45, and 19 kDa in the lipid-laden plaques. Lipid accumulation in the plaque was also associated with a loss of smooth muscle cells, the cellular source of the collagen fibers. The remaining smooth muscle cells showed increased collagen synthesis, although it was insufficient to counterbalance collagen degradation and cell loss. Thus, we have obtained direct evidence that hypercholesterolemia is accompanied by enhanced local collagen degradation, which is potentially responsible for plaque weakening.