Urinary peptides in Rett syndrome.

Rett syndrome is a neuro-developmental disorder related to autistic behavior. Persons with autism have previously been found to have hyperpeptiduria. We here report a significantly higher level of peptides in the first fasting morning urine from 53 girls with Rett syndrome (both classical and congenital) compared with 53 healthy girls. This elevation in urinary peptides was similar to that in 35 girls with infantile autism. As in persons with autism, the individual levels of urinary peptides in the Rett syndrome group varied, and about a fifth were within the normal range. Levels of peptides were lower in girls with classic Rett syndrome than in girls with congenital Rett syndrome. This may be due to different etiological causes or to active and stagnant phases of the disease. Urine from girls with Rett syndrome was found to have higher frequency and higher levels of some urinary peptides that may cause inhibition of brain maturation and epilepsy

Elemental diets in the repair of small intestinal damage.

We have investigated the possible benefits of elemental diets, especially a diet supplemented with L-glutamine, on maintenance of intestinal absorptive function in rat small intestine damaged by 5-fluorouracil. Although a standard rat diet sustained better body growth in control rats, each of the elemental diets and the diet containing intact casein in place of hydrolyzed casein was beneficial in promoting less body weight loss during the 3 d after 5-fluorouracil injection. The same significant benefit was seen in absorptive activity measured in small intestine in vitro 3 d after the cytotoxic injury. Glutamine supplementation, however, did not confer any significant advantages, although it did cause significant elevation of muscle glutamine pools. This elevation was substantially less than the corresponding increase in muscle glycine content after dietary supplementation with glycine.

Some considerations regarding the use of amino acid racemization in human dentine as an indicator of age at death.

An HPLC method is described for simultaneously obtaining the enantiomeric ratio of three amino acids (aspartic acid, serine, and glutamic acid) from dental collagen, with a view to using this information for estimating age at death. Results are reported from a sample of twenty three known age modern teeth, six known age 19th C. AD teeth, and two unknown age Romano-British teeth. It was found (as expected) that all three D/L ratios changed significantly with chronological age. Standard calibration techniques were used to estimate ages for the six 19th C. AD specimens from regression equations estimated from the modern specimens, and also to predict (for the first time) the error associated with such estimates. Errors using aspartic acid were found to be similar to those obtained by other methods of age estimation from dental evidence, serine, and glutamic acid providing much poorer age estimates. Additionally, a systematic difference in the age-enantiomeric ratio relationship was observed between modern and older dental samples. It is concluded that there is some fundamental difference in the observed enantiomeric ratios between modern teeth and older samples, possibly as a result of the chemical alteration of the dental proteins.

The induction of the alpha-1-adrenoceptor signal transduction system on human melanocytes.

Human melanocytes established in MCDB-153 culture medium do not express alpha 1-, beta 1-, beta-2 adrenoceptors without extracellular stimulation. The addition of 50 x 10-9 M norepinephrine to the medium causes a time-dependent induction of alpha-1-adrenoceptors with 4.278 receptors/melanocyte after 24 h. Under the same experimental conditions, the dendricity of melanocytes as well as melanogenesis was unaffected over 60 h. Since keratinocytes hold the full capacity for catecholamine biosynthesis but melanocytes lack this system, the secretion of catecholamines from keratinocytes appears to be of critical importance to the alpha-1-adrenoceptor in melanocytes, underlining the symbiosis of both cells in the epidermal unit.

A specific tetrahydrobiopterin binding domain on tyrosinase controls melanogenesis.

(6R)5,6,7,8-tetrahydrobiopterin (6-BH4) directly regulates tyrosinase activity by specifically binding to a putative 13 amino acid domain. This domain has sequence homology to 6-BH4 binding sites already identified on phenylalanine hydroxylase and 4a-carbinolamine dehydratase. Furthermore, this binding sequence appears to have been conserved during the evolution of tyrosinase as it has also been identified in the frog, mouse and human enzymes. 6-BH4 controls tyrosinase activity by an uncompetitive mechanism requiring the presence of L-tyrosine for effective down-regulation. When L-dopa is substrate, 6-BH4 does not inhibit the enzyme implicating separate binding sites for L-dopa and L-tyrosine on tyrosinase. Dihydropterin and 6-biopterin, the oxidation products of 6-BH4, do not inhibit tyrosinase significantly, indicating that melanin biosynthesis is controlled by a 6-BH4/6-biopterin redox-switch mechanism which can be initiated by photo-oxidation of 6-BH4.

Intestinal absorption of the intact peptide carnosine in man, and comparison with intestinal permeability to lactulose.

1. Healthy humans ingested the dipeptide carnosine (L-beta-alanyl-L-histidine). Their plasma levels and urinary outputs of carnosine and beta-alanine were monitored over the following 5 h. 2. Large amounts of intact carnosine (up to 14% of the ingested dose) were recovered in the urine over the 5 h after ingestion. However, carnosine was undetectable in the plasma unless precautions were taken to inhibit blood carnosinase activity ex vivo during and after blood collection. 3. The amount of carnosine recovered in urine varied substantially between subjects. It correlated negatively with carnosinase enzymic activity in the plasma. Highest carnosinase activities were observed in those subjects who regularly underwent physical training. 4. Urinary recovery of the disaccharide lactulose also varied considerably between subjects, but was substantially lower than that of carnosine. There was no significant correlation between the recoveries of carnosine and lactulose. 5. When lactulose was ingested with a hypertonic solution, the urinary recovery of lactulose was generally increased. When carnosine was ingested with a hypertonic solution, the urinary recovery of carnosine was reduced: hence the paracellular route probably is not dominant for absorption of intact carnosine. 6. Intact carnosine must have crossed the intestine to an extent much greater than hitherto recognized. Rapid post-absorptive hydrolysis is a severe obstacle to quantification of intact peptide absorption.

Simple, optimized liquid-chromatographic method for measuring total hydroxyproline in urine evaluated.

We have optimized a method for measuring total hydroxyproline (HYP) in urine by HPLC after release from urinary peptides by solid-phase hydrolysis on Dowex 50W x 8 ion-exchange resin. The HYP was derivatized with 4-chlor-7-nitrobenzo-2-oxa-1,3-diazole, and excess reagent was removed with the use of a 100-mg C18 Bond-Elut cartridge. The HYP derivative was separated isocratically at 30 degrees C on a 250 x 4.6 mm reversed-phase column containing 5-microns particles of Spherisorb S5 ODS-2, with S-carboxymethylcysteine as internal standard. Total assay time was 14 min. The standard curve for the method was linear from the detection limit for HYP, 3.6 mumol/L, to 10 mmol/L. The between-batch CV was less than 5.1% and the mean analytical recovery of HYP was 95% +/- 1.4%. Comparison with a commercially available colorimetric method showed good correlation: y = 1.158x + 19.76 mumol/L (Syx = 74, n = 120), but HPLC results were 15% higher, probably from incomplete hydrolysis with the colorimetric method. This method offers a considerable improvement in assay time, specificity, sensitivity, precision, and cost compared with the colorimetric method.

Treatment of encainide proarrhythmia with hypertonic saline.

We report a case of successful treatment of encainide-induced ventricular tachycardia with 3% hypertonic saline. To our knowledge, no other report exists in the literature of this treatment for proarrhythmic ventricular tachycardia from a type 1C agent. Metabolic consequences of the treatment included severe hypernatremia, hyperosmolarity, hypocalcemia, and hypophosphatemia, which were reversible over 24 hours. In spite of the risks, treatment of incessant ventricular tachycardia induced by type 1C agents with hypertonic saline may be life saving.

Sodium loading treatment for amphotericin B-induced nephrotoxicity.

The increased frequency and duration of antifungal treatment with amphotericin B in immunocompromised patients has stimulated a great deal of research into the mechanisms of its nephrotoxic effects and treatment modalities designed to attenuate these effects. A review of amphotericin B-induced nephrotoxicity, the underlying pathophysiologic mechanisms, and the role of salt loading as a means of minimizing renal impairment are described. Both animal and human studies regarding the efficacy of sodium loading are presented as well as a case report describing the use of salt supplementation over a prolonged course of therapy.

Is there circadian variation in villus height in rat small intestine?

Measurements of villus height, crypt depth and mucosal thickness were made for each of duodenum, jejunum and ileum of rat small intestine from animals on both unrestricted and restricted feeding regimens. Contrary to a previous report, there was no evidence for consistent or synchronized diurnal variation in villus height at any region of small intestine on any of the feeding regimes. Likewise, no diurnal variation was seen in crypt depth or mucosal thickness. Variation in villus cell numbers, therefore, cannot account for previously reported diurnal variation in absorptive and digestive activities in rat small intestine. The present results support the hypothesis that the stimulus for exfoliation is probably cell recruitment and migration up the villi.

Gastrointestinal absorption of intact proteins.

There is now no reasonable doubt that small quantities of intact proteins do cross the gastrointestinal tract in animals and adult humans, and that this is a physiologically normal process required for antigen sampling by subepithelial immune tissue in the gut. It is too small to be nutritionally significant in terms of gross acquisition of amino-nitrogen, but since it has important implications relating to dietary composition it must receive consideration from nutritionists. The process of intact protein absorption occurs without eliciting harmful consequences for most individuals, but it appears likely that a small number of people absorbing these "normal" amounts may react idiosyncratically; also, some individuals may absorb excessive amounts, and they may suffer clinically significant consequences. Likewise, individuals with diminished absorption of intact protein may be at risk. Normal absorption probably occurs predominantly by transcellular endocytosis with some possible contribution by a route between cells; increased net entry of protein to the circulation may reflect (a) increased paracellular (intercellular) passage, (b) increased transcellular passage, and/or (c) decreased lysosomal proteolysis. Tests to distinguish among these possibilities are strongly desirable. Intact protein absorption may be involved in the pathogenesis of inflammatory bowel disease, "food allergies," and other diseases, including even major psychiatric disorders, but the current evidence is mainly indirect and suggestive. Great caution and careful objective studies are needed to establish whether such relationships with disease do exist and to unravel the underlying basic physiological mechanisms. Now that interest has developed in the assessment of intestinal permeability to small- and medium-sized molecules, it is hoped that equally simple methods for studying macromolecular permeability will be developed and applied. Therapeutic methods for enhancing intact polypeptide absorption would be valuable for vaccine and peptide drug administration by the oral route. Therapeutic reduction of the process may be relevant in food-sensitive patients.

Alternate models for shared carriers or a single maturing carrier in hexose uptake into rabbit jejunum in vitro.

The uptake (tissue accumulation) of three hexoses into rabbit jejunum was measured in a flux chamber in conditions of effective stirring. Glucose uptake was inhibited by galactose or 3-O-methylglucose: 1-40 mM galactose caused a progressive decline in glucose uptake; 1-5 mM 3-O-methylglucose inhibited glucose uptake but higher concentrations of 3-O-methylglucose had no further effect. When 1-40 mM 3-O-methylglucose was added to glucose plus galactose there was a further decrease in the uptake of glucose; adding 1-40 mM galactose to glucose plus 3-O-methylglucose also produced a decrease in glucose uptake. Both glucose and 3-O-methylglucose inhibited uptake of galactose but the pattern of inhibition varied between the two sugars. The uptake of 3-O-methylglucose was also inhibited by glucose and by galactose, but the uptake of 3-O-methylglucose in the presence of either galactose or glucose was no further reduced by adding the third hexose. Graphical analysis and analysis by non-linear regression both showed that neither the single Michaelis-Menten function, nor the single Michaelis-Menten-plus-competitive-inhibition function was appropriate for any of these data. The results are consistent with the hypothesis that either there are multiple (at least three) intestinal carriers for hexoses; alternatively that there is a single carrier whose transport properties for the three hexoses change differentially during cell maturation and migration up the villus.

A comparison of the structural integrity of several commonly used preparations of rat small intestine in vitro.

The structural integrities of various preparations of rat small intestine for the study of absorption in vitro have been compared after incubation or perfusion. Perfused intestines removed from anaesthetized rats, and thus never deprived of a supply of oxygen, maintain their structural integrity even after perfusion for 1 h provided that a Krebs-Henseleit bicarbonate perfusate is used. However, intestines removed from freshly killed rats show severe villus disruption and oedema after perfusion for only 20 min. Extensive damage to both crypts and villi is observed in everted sacs of small intestine incubated for 20 min, regardless of the buffer system used. Intestinal rings show damage at the tips of the villi after incubation for 2 min, but otherwise remain morphologically intact; this damage is progressive with time. It is concluded that the exact mode of preparation of intestinal tissue is critical for preservation of structural and functional integrity and that this is especially important in quantitative studies on transport processes. Further, it is recommended that routine monitoring of the integrity of intestinal preparations in vitro is desirable and that histological assessment is an appropriate technique.

A comparison of internal and external standardization in amino acid analysis.

To assess quantitatively the merits of internal standardization, an amino acid mixture of known composition has been analyzed by conventional automated amino acid analysis before and after being subjected to total acid hydrolysis. Both the precision (reproducibility) and the accuracy (lack of bias) were generally, but not invariably, greatly enhanced by the use of internal standardization with norleucine as opposed to external standardization. Incorporation of internal standards into samples is strongly recommended.

Cysteine: a potential source of error in amino acid analysis of mercaptoethane sulfonic or hydrochloric acid hydrolysates of proteins and peptides.

Hydrolysis of proteins and peptides with mercaptoethane sulfonic acid is liable to produce overestimation of the proline content owing to the production of ninhydrin-positive material (probably cysteine) which coelutes with proline on many ion-exchange analytical systems. A similar error occurs with HCl hydrolysis (especially in the presence of mercaptoethanol or thioglycollic acid) if care is not taken to oxidize cysteine during reconstitution of the hydrolysate before amino acid analysis.

Can elemental diets reduce the intestinal toxicity of 5-fluorouracil?

In order to test the hypothesis that elemental diets protect the intestinal mucosa against 5-fluorouracil toxicity, we have estimated water absorption, cytoplasmic peptide hydrolase activities, and mucosal DNA contents in isolated intestines from rats fed on one of three elemental diets or a standard diet and injected with 5-fluorouracil. Water absorption rates were significantly increased when rats, not injected with 5-fluorouracil, were fed on Flexical or Vivonex-HN. However, water absorption was severely impaired 3 days after administration of 5-fluorouracil and none of the elemental diets alleviated this impairment of absorption. Cytoplasmic peptide hydrolase activities and mucosal DNA contents were also severely decreased after 5-fluorouracil injection, the changes observed being independent of the diet. Also, none of the elemental diets reduced the body weight losses observed after 5-fluorouracil administration. Although the effects of the sodium salt of 5-fluorouracil on body weight and food intakes were much less severe than those of the Tris salt the two salts had identical effects on intestinal absorption even when an elemental diet was used. Although there was a smaller incidence of diarrhea after 5-fluorouracil administration when the rats had been fed on one of the elemental diets, diet B, the results do not support the suggestion that elemental diets might reduce the intestinal toxicity of 5-fluorouracil.