Effects of nitrous oxide-induced inactivation of cobalamin on methionine and S-adenosylmethionine metabolism in the rat.

Inhalation of nitrous oxidises cobalamin and, in turn, inactivates methionine synthetase which forms methionine from homocysteine and which requires cob[I]alamin as a co-factor. This study was planned to determine the effect of virtual cessation of methionine synthesis via a cobalamin-dependent pathway, on tissue levels of methionine, S-adenosylmethionine and on related enzymes. The level of methionine in liver fell initially after exposure to N2O but was restored to pre-N2O levels after 6 days despite continuing N2O exposure. Brain methionine fell within 12 h of N2O exposure but the fall was not significant. The restoration of methionine levels is accompanied by an increase in activity of betaine homocysteine methyltransferase in liver but this enzyme was not detected in brain. The activity of methionine synthetase remained very low in both liver and brain as long as N2O inhalation was continued. There was an initial rise in liver S-adenosylmethionine levels followed by a steady fall to 40% of its initial level after 11 days of N2O exposure. However, there was no change in the level of S-adenosylmethionine in brain during this period. The data indicate that either brain meets its requirement by increased methionine uptake from plasma or that there are alternate pathways in brain for methionine synthesis other than those requiring a cobalamin coenzyme.

Molecular identification of binding sites for calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide (IAPP) in mammalian lung: species variation and binding of truncated CGRP and IAPP.

We have investigated the binding of rat [125I]islet amyloid polypeptide (IAPP) and [125I]calcitonin gene-related peptide (CGRP) to lung membranes from the rat, rabbit, and bull and have characterized the mol wt (M(r)) of the binding site for each ligand by chemical crosslinking. Results imply the existence of three distinct types of binding site demonstrated by both [125I]CGRP and [125I]IAPP in each of the three species investigated. These were differentiated by the relative potencies of displacement by rat CGRP, human CGRP-(8-37), rat IAPP, and the rat IAPP fragments IAPP-(8-37), IAPP-(12-37), IAPP-(25-37), and IAPP-(28-37). High affinity binding sites were identified for [125I]CGRP [rat Ki, 0.119 +/- 0.027 nM (n = 6); rabbit Ki, 0.944 +/- 0.075 nM (n = 6); bull Ki, 0.20 +/- 0.031 nM (n = 6)], and CGRP-(8-37) was found to displace [125I]CGRP in all species [rat Ki, 6.63 +/- 0.91 nM (n = 6); rabbit Ki, 22.70 +/- 3.79 nM (n = 6); bovine Ki, 26.9 +/- 0.21 nM (n = 3)]. Compared to CGRP-(8-37), displacement by IAPP also showed varying affinities that were similar to that of CGRP-(8-37) (rat), lower than that of CGRP-(8-37) (rabbit), or higher than that of CGRP-(8-37) (bull). Truncation of IAPP caused large parallel decreases in its affinity for [125I]CGRP in the rabbit and bull by the loss of residues 1-8 (rabbit) and 1-12 (bull), but was not as pronounced in the rat. [125I]IAPP demonstrated high affinity binding in each species [rat Ki, 5.86 +/- 0.86 nM (n = 6); rabbit Ki, 18.72 +/- 2.90 nM (n = 6); bull Ki, 1.97 +/- 0.40 nM (n = 6)]. Truncation of IAPP caused a reduction of its affinity for [125I]IAPP in all species by the loss of residues 1-28. Chemical cross-linking analysis indicated binding of both ligands to sites of 64,000 M(r) in the rat and 50,500 and 51,000 M(r) in the rabbit and bull, respectively. In addition, [125I]IAPP bound to to a site of 100,000 M(r) in the rat. [125I]CGRP and [125I]IAPP binding were reduced in the presence of guanosine 5-o-(3-Thiotriphosphate) in all species, indicating an association with G-proteins. This study implies the existence of CGRP/IAPP-binding sites in the lungs of these species that show varying and complex patterns of displacement by CGRP, IAPP, and their fragments.

Combined hepatic and renal transplantation in primary hyperoxaluria type I: clinical report of nine cases.

PURPOSE AND PATIENTS AND METHODS: The purpose of this article is to report the experience of three centers with combined hepatic and renal transplantation for pyridoxine-resistant primary hyperoxaluria type I (alanine:glyoxylate aminotransferase [EC 2.6.1.44] deficiency), with particular emphasis on the selection criteria and timing of the operation. Nine patients with this inherited disease were treated by combined hepatic and renal transplantation. The former replaces the enzyme-deficient organ while the latter replaces the functionally affected organ. RESULTS: One patient with gross systemic oxalosis died in the immediate postoperative period and another died 8 weeks postoperatively of a generalized cytomegalovirus infection, having shown evidence of biochemical correction. One patient with particularly severe osteodystrophy at the time of the operation died 14 months postoperatively from renal failure due to progressive calcium oxalate nephrocalcinosis involving the transplanted kidney, plus thromboembolic disease. He also had very extensive systemic oxalosis. An additional patient with severe osteodystrophy died 9 months postoperatively. One patient developed hyper-rejection of the kidney and died later of gastrointestinal hemorrhage. The four long-term survivors (22 to 38 months) have remained asymptomatic from the standpoint of their renal disease, with resolution of any manifestations of systemic oxalosis that they may have had. They are either employed or continuing their education. CONCLUSIONS: A prolonged period of end-stage renal failure treated by dialysis regimens that are suitable for non-hyperoxaluric renal failure and extensive systemic oxalosis, particularly oxalotic osteodystrophy, are poor prognostic features. We propose that hepatic transplantation should be considered as definitive treatment before end-stage renal failure develops. This should be supplemented by renal transplantation with vigorous pre- and perioperative hemodialysis to deplete the body stores of oxalate. Although some authorities would reserve hepatic transplantation for patients in whom renal transplantation has failed, we suggest that combined liver and kidney transplantation is appropriate in patients who have never had a renal graft. Furthermore, the time has come to consider hepatic transplantation before any irreversible renal damage has occurred in these patients.

Vitamin B12 neuropathy is not due to failure to methylate myelin basic protein.

It has been proposed that the biochemical lesion in subacute combined degeneration of the cord due to vitamin B12 deficiency, is impaired methylation of residue 107 (arginine) in myelin basic protein. We have examined myelin basic protein in brains of rats in which vitamin B12 was inactivated by exposure to nitrous oxide for up to 7 days. In addition brains of fruit bats in which vitamin B12 neuropathy had been produced by feeding washed, and hence vitamin B12-free fruit, were examined. There was no difference in the methylation of arginine 107 in myelin basic protein in these animals as compared to healthy control animals. Rats given an inhibitor of transmethylation reactions (cycloleucine) showed the expected fall in methylation of myelin basic protein.

Studies on the composition of urinary glycosaminoglycans and oligosaccharides in patients with mucopolysaccharidoses who were receiving fibroblast transplants.

This communication reports studies on the composition of the urinary glycosaminoglycans and oligosaccharides in mucopolysaccharidosis patients who were being treated by fibroblast transplantation. The urinary glycosaminoglycans were precipitated with 9-aminoacridine, the oligosaccharides remaining in solution. Both fractions were further subfractionated by gel filtration. The glycosaminoglycan subfractions were examined for their content of iduronic acid, glucuronic acid, galactosamine and glucosamine. We found no changes in these parameters in a patient who had been treated by repeated fibroblast transplantations over the course of 4 1/2 years. The amino sugar composition of the oligosaccharide fraction was examined and shown to be unchanged. We also found no changes in the degree of sulphation of the urinary glycosaminoglycans specifically related to the transplant in four patients with Hurler disease and two with Hunter disease. We conclude that fibroblast transplantation does not produce detectable changes in the carbohydrate content or degree of sulphation of the urinary glycosaminoglycans and oligosaccharides.

Renal transplantation in primary hyperoxaluria.

The increased production and excretion of oxalate in primary hyperoxaluria causes urolithiasis, nephrocalcinosis with renal failure, and systemic oxalosis. Systemic oxalosis occurs late in the course of the disease when there is both oxalate retention and increased oxalate synthesis. The uraemia can be controlled by conventional haemodialysis or peritoneal dialysis but treatment cannot usually keep up with accelerated rate of oxalate production, and dialysed patients develop systemic oxalosis. Most attempts to treat primary hyperoxaluria by renal transplantation have been unsuccessful because of rapid recurrence of nephrocalcinosis with uraemia and systemic oxalosis. Dynamic studies of overall oxalate metabolism in vivo have shown that the renal retention factor becomes a major determinant of oxalosis when the GFR decreases to less than 25 ml min-1 1.73 m-2. We conclude provisionally that vigorous haemodialysis should be begun and transplantation arranged when the GFR reaches this level. Such early transplantation with vigorous perioperative haemodialysis and a large perioperative diuresis of water gives good immediate graft function and oxalate mobilisation from the miscible oxalate pool. The longer term outlook is then influenced more by the factors which determine the success of renal transplantation in non-hyperoxaluric patients.

Transport of methionine and proline by rat liver slices and the effect of certain hormones.

1. Transport characteristics of l-methionine and l-proline in rat liver slices in vitro were studied. 2. Intracellular concentration gradients for methionine were obtained. 3. Methionine uptake was inhibited by iodoacetate, dinitrophenol, Na(+)-free media and also by glycine, lysine, cysteine and dithiothreitol but not by alpha-aminoisobutyrate. 4. The rate of methionine metabolism in the slice was slow. 5. Puromycin inhibited methionine incorporation into protein, but not methionine uptake. 6. Methionine inhibited the transport of alpha-aminoisobutyrate but not of cystine. 7. Efflux and exchange diffusion of methionine was studied. 8. Amino acid transport in rat liver slices was not affected by thyroidectomy. 9. Addition of insulin, glucagon, adrenaline or cortisol did not affect the transport of methionine. 10. Addition of 6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate increased methionine transport after a 120min incubation period in some experiments. 11. Studies of l-proline transport were invalidated because of the rapid evolution of CO(2) from the substrate.

Low Dose D-penicillamine in cystinuria.

(1) A single dose of D-penicillamine (not more than 750 mg) taken at 2200 h, together with a prescribed fluid intake of two to three litres during the waking hours, without extra drinks at night, is sufficient to keep the concentration of the urinary cystine below the saturating concentration of urine throughout the 24 h period. (2) This regime does not materially reduce the total 24 h excretion of cystine. (3) The effect of D-penicillamine is mainly seen in the urine excreted between 0200 and 0800 h. (4) This regime is provisionally recommended for the prevention of cystine stone recurrence but not for stone dissolution. Larger total amounts of D-penicillamine given in divided doses are still recommended for the latter purpose.

Sequential studies of oxalate dynamics in primary hyperoxaluria.

We have measured the total plasma clearance, renal clearance and equilibrium distribution volume of [14C]oxalate, and the urinary oxalate excretion rate and plasma oxalate levels at approximately 6 months intervals for up to 2.5 years in five patients with primary hyperoxaluria. The renal clearance and distribution volumes of [99mTc]DTPA (diethylenetriaminepenta-acetate) were measured simultaneously to provide estimates of glomerular filtration rate (GFR) and extracellular fluid volume (ECF). The same measurements were made on each of five normal volunteers. Clearances and distribution volumes were measured with a modified single injection technique. The oxalate clearance was two to three times the simultaneously measured GFR in the patients and control subjects. The renal clearance of oxalate was less than the total plasma clearance in the patients. The oxalate distribution volume was approximately 1.5 times the ECF in both the patients and controls. Only small changes were observed over a 2.5 years period in these particular patients. The plasma oxalate concentration was derived from the urinary oxalate excretion rate and the plasma [14C]oxalate clearance. It was raised in the patients. The oxalate removal rate was derived from the total plasma clearance and the plasma oxalate concentration.

Screening for urinary oligosaccharides: a quantitative procedure.

A method for screening urine for abnormalities in glycoconjugate excretion is presented. An oligosaccharide-containing fraction is isolated from urine by gel chromatography and the carbohydrate-containing components in this fraction are degraded to monosaccharides, suitable derivatives of which are analyzed by gas-liquid chromatography. A pattern which characterizes the monosaccharides originating in complex carbohydrates in urine is thus obtained. Data obtained by applying this method to normal subjects in various age groups are presented and the applicability of the method and its limitations are illustrated by the use of examples from patients with previously diagnosed disorders of glycoconjugate metabolism.

Screening for urinary oligosaccharides: application to a mentally handicapped population and to patients with some other diseases.

The method for screening urinary oligosaccharides described in the accompanying paper [Clin. Chem. 24, 669 (1978)] has been applied to a large group of mentally handicapped patients as well as to patients with cystic fibrosis, malignant disease, and schizophrenia. The cause of the mental handicap was unknown in most cases. The primary object of the study was to investigate a possible link between some diseases and urinary complex carbohydrates. Our results show that abnormalities that are detectable in this way do not contribute significantly to the overall incidence of mental handicap, nor are they expressed in the other diseases studied.

Comparison of human myofibrillar protein catabolic rate derived from 3-methylhistidine excretion with synthetic rate from muscle biopsies during L-[alpha-15N]lysine infusion.

1. Urine was collected in five healthy men over 10--14 days, with fasting blood samples on days 1, 5 and 10, whilst they consumed a standard creatine-free diet, which was quantitatively related to their body surface area. 2. The urinary excretion of 3-methylhistidine fell to a plateau by day 5 in all subjects. Myofibrillar protein catabolic rate calculated from the mean value of 3-methylhistidine excretion from day 5 to day 10 averaged 1.21 g day-1 kg-1 body weight. The average turnover of muscle myofibrillar protein was calculated to be 2.16%/day. 3. From a previous study using continuous intravenous infusion of L-[alpha-15N]lysine with serial muscle biopsies on the same subjects, the mean myofibrillar protein synthetic rate was calculated to be 0.82 g day-1 kg-1 body weight, and the mean turnover rate was 1.47%/day of total muscle myofibrillar protein. 4. The estimations of myofibrillar protein turnover rate derived from the two methods are compared and the differences discussed.

Combined iminoglycinuria and cystine- and dibasic aminoaciduria in patients with propionic acidaemia and 3-methylcrotonylglycinuria.

Urinary amino acids have been determined in six patients with propionic acidaemia, one of whom also showed 3-methylcrotonylglycinuria. Two patients, including the subject with 3-methylcrotonylglycinuria, showed a gross aminoaciduria with features of both cystinuria and iminoglycinuria. We suggest a defect in certain amino acid transport systems in some patients with these disorders.

A new variant form of phenylketonuria.

We report a family (parents and two sets of twin girls) in which the propositus presented as a case of phenylketonuria with a somewhat less severe degree of phenylalanine intolerance than occurs in patients with classical phenylketonuria and whose phenylalanine tolerance was further impaired by giving cotrimoxazole. The trimethoprim component of cotrimoxazole reduces the phenylalanine tolerance of normal subjects but does not augment the degree of phenylalanine intolerance in patients with classical phenylketonuria. The results of the present study of the phenylalanine tolerance and the way in which cotrimoxazole modifies it in the members of this family are compatible with the segregation of an abnormal gene which causes a previously unrecognized type of phenylketonuria when it is present in the homozygous state. The possible relationship of this to other inherited biochemical lesions of the phenylalanine hydroxylase system is briefly discussed.