Mechanical and metabolic viability of a placental perfusion system in vitro under oxygenated and anoxic conditions.

In vitro dual circuit perfusion of the placenta with well-oxygenated medium results in the continuous and stable consumption of oxygen and glucose over a 2-h perfusion period. This is reflected in a stable production of lactate and an energy charge which is higher at the end of the perfusion period than that seen in fresh placental tissue immediately after vaginal delivery. Anoxic perfusion causes an increase in glucose consumption which is more than twofold higher than that seen in the oxygenated perfusion, resulting finally in placental uptake of glucose not only from the maternal but also from the fetal circulation. Lactate production is increased during the anoxic perfusion, while the final tissue energy charge value lies between the values observed for fresh tissue and for the oxygenated perfusion. The shift to anaerobic metabolism shown by placental tissue in anoxic conditions enables continued functioning of the tissue over the 2-h perfusion period but it appears that under anoxic conditions the tissue may incur an energy debt not observed in oxygenated perfusions.

Creatine metabolism during metabolic perturbations in patients with organic acidurias.

Creatine excretion was measured in two patients with methylmalonic aciduria and two patients with 3-hydroxy-3-methylglutaric aciduria. During periods of metabolic decompensation the creatine/creatinine ratio increased and fell during recovery. Prolonged periods of metabolic decompensation may result in the loss of a large proportion of the creatine pool. In one study, measurements of total daily urinary output of metabolites demonstrated that the absolute creatine excretion followed a similar qualitative pattern to the creatine/creatinine ratio. However, apparent fluctuations in methylmalonate excretion when expressed as methylmalonate/creatinine ratio were absent when absolute methylmalonate excretion was calculated. The increased creatine excretion during metabolic perturbations may result from loss from creatine containing tissues such as muscle and may represent an underlying defect in energy metabolism. Alternatively creatine transport may be disrupted by accompanying acidosis. The use of metabolite/creatinine ratios as a measure of metabolite excretion rates during metabolic decompensation whilst qualitatively sound may need a re-appraisal.

Carnitine therapy and metabolism in the disorders of propionyl-CoA metabolism studied using 1H-NMR spectroscopy.

1H-NMR spectroscopy has been used to study metabolic perturbations in patients with disorders of propionyl-CoA metabolism during the administration of oral and intravenous L-carnitine. The administration of L-carnitine either in the form of a challenge or as a therapeutic measure resulted in an increased excretion of propionylcarnitine, consistent with the removal of accumulated intramitochondrial propionyl-CoA esters. Additionally, during the therapeutic administration of L-carnitine excretion of acetylcarnitine occurred, coincident with an improvement in clinical condition and confirming the intracellular propionyl-CoA depletion. An additional benefit from the formation of acylcarnitines may be an accompanying intracellular alkalinisation.

Measurement of L-carnitine and acylcarnitines in body fluids and tissues in children and in adults.

We have developed a reliable and validated radio-enzymatic method for the assay of L-carnitine and acylcarnitines, using a modification of existing methods. The sensitivity of the assay is 10 mumol/l using 10 microliters of plasma or urine. It is also suitable for measurements of carnitine in a 10 mg sample of liver or muscle obtained by percutaneous biopsy. The use of N-ethylmaleimide in the reaction mixture together with an excess of [1-14C]acetyl CoA ensures that the reaction proceeds to completion and a linear response is obtained. Using this method control ranges have been established for plasma and urine carnitine concentrations in healthy children and adults, and for the carnitine content of liver and muscle in adults. No significant difference was found between fasting and post-prandial plasma carnitine levels. An age-related increase was found in urinary total carnitine and acylcarnitine concentration throughout childhood. These data provide a reliable basis for studies of patients with abnormal carnitine and acylcarnitine metabolism, distribution and excretion.

Urinary C6-C12 dicarboxylic acylcarnitines in Reye's syndrome.

C6-C12 dicarboxylic acylcarnitines have been identified for the first time in urine from a 2-year-old girl presenting with Reye's syndrome. The acylcarnitines were extracted by ion-exchange chromatography and analysed, both underivatised and as methyl esters using high-resolution fast-atom-bombardment mass spectrometry and B/E-linked scanning. The acylcarnitines were quantified by capillary gas chromatography of the acids extracted after hydrolysis of the acylcarnitine esters. Dodecandioylcarnitine was present in the highest concentration (35.9 mmol/mol creatinine) which exceeded the urinary free dodecandioic acid concentration. The adipic, suberic and sebacic acylcarnitine concentrations were less than 10% of the respective free acid concentrations. It is possible that beta-oxidation of dicarboxylic acids is partially inhibited in Reye's syndrome leading to accumulation of precursor dodecandioyl CoA which is metabolised to dodecandioylcarnitine. The accumulation of these metabolic intermediates may be significant in the pathogenesis of Reye's syndrome.

Maternal alloimmunization to HLA, platelet and granulocyte-specific antigens during pregnancy: its influence on cord blood granulocyte and platelet counts.

In order to obtain an estimate of the frequency of platelet-specific and granulocyte-specific antibodies and of the effect of such antibodies on the platelet count and granulocyte count of the newborn infant, serum from 147 women in their second or subsequent pregnancies was tested. No platelet-specific antibodies were found but 29 of the women had granulocyte-specific antibodies and the corresponding infants had granulocyte counts which were significantly lower than those of infants without antibodies. HLA antibodies were found in the sera of 57 women but were not associated with diminished platelet or granulocyte counts in the corresponding infants. Maternal granulocyte antibodies may be an underestimated contributory factor in the pathogenesis of neonatal neutropenia.

Deficient fumarase activity in an infant with fumaricacidemia and its distribution between the different forms of the enzyme seen on isoelectric focusing.

A male infant, whose parents were first cousins, presented at 6 mo of age with hypotonia, microcephaly, and delayed development. He was found to have large amounts of fumaric and succinic acids present in the urine. In lysed cultured skin-fibroblast preparations, the activity of fumarase was found to be 22.7% of that in controls. Cell fractionation by homogenization and by digitonin treatment indicated that the residual activity in the cells of the patient was primarily located in the mitochondrial fraction rather than in the cytosolic fraction. Isoelectric focusing of fibroblast extracts showed that six bands of fumarase activity were discernible in control cell lines, two of them cytosolic with pI's of 5.53 and 5.60 and four of them mitochondrial with a pI of 5.65-6.8. In contrast, isoelectric focusing of fibroblast extracts from the fumarase-deficient patient showed only a single band of activity with a pI corresponding to the mitochondrial type seen in the controls. Immunoprecipitation of proteins with rabbit antifumarase antibody in (35S)-methionine-labeled fibroblasts indicated that a protein of correct size (Mr = 44,000 daltons) corresponding to fumarase was synthesized in similar amounts in both the patients and controls. It is proposed that in the patient's cells a single active species of fumarase that is mitochondrial in location is synthesized. Since it is known that mitochondrial and cytosolic fumarases are encoded by the same gene but differ slightly in amino acid sequence, it is possible that a point mutation might explain these findings.

Urinary excretion of l-carnitine and acylcarnitines by patients with disorders of organic acid metabolism: evidence for secondary insufficiency of l-carnitine.

Concentrations of l-carnitine and acylcarnitines have been determined in urine from patients with disorders of organic acid metabolism associated with an intramitochondrial accumulation of acyl-CoA intermediates. These included propionic acidemia, methylmalonic aciduria, isovaleric acidemia, multicarboxylase deficiency, 3-hydroxy-3-methylglutaric aciduria, methylacetoacetyl-CoA thiolase deficiency, and various dicarboxylic acidurias including glutaric aciduria, medium-chain acyl-CoA dehydrogenase deficiency, and multiple acyl-CoA dehydrogenase deficiency. In all cases, concentrations of acylcarnitines were greatly increased above normal with free carnitine concentrations ranging from undetectable to supranormal values. The ratios of acylcarnitine/carnitine were elevated above the normal value of 2.0 +/- 1.1. l-Carnitine was given to three of these patients; in each case, concentrations of plasma and urine carnitines increased accompanied by a marked increase in concentrations of short-chain acylcarnitines. These acylcarnitines have been examined using fast atom bombardment mass spectrometry in some of these diseases and have been shown to be propionylcarnitine in methylmalonic aciduria and propionic acidemia, isovalerylcarnitine in isovaleric acidemia, and hexanoylcarnitine and octanoylcarnitine in medium-chain acyl-CoA dehydrogenase deficiency. The excretion of these acylcarnitines is compatible with the known accumulation of the corresponding acyl-CoA esters in these diseases. In this group of disorders, the increased acylcarnitine/carnitine ratio in urine and plasma indicates an imbalance of mitochondrial mass action homeostasis and, hence, of acyl-CoA/CoA ratios. Despite naturally occurring attempts to increase endogeneous l-carnitine biosynthesis, there is insufficient carnitine available to restore the mass action ratio as demonstrated by the further increase in acylcarnitine excretion when patients were given oral l-carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)

Permeability of the sheep placenta to unmetabolized polar non-electrolytes.

1. The rate of appearance in uterine venous blood of radioactively labelled, polar, non-electrolytes has been measured following their injection into the foetal circulation in the chronically catheterized sheep near term. Uterine blood flow was measured by an antipyrine technique. 2. Estimates of a placental permeability constant not corrected for area were 61-2 +/- 5-5 ml. min-1 (mean +/- S.E. of mean) for [14C] urea, 1-85 +/- 0-16 for [14C]erythritol and 0-21 +/- 0-03 for [3H]mannitol. Results are also presented for [14C]ethylene glycol, [14C]L-glucose, [14C]mannitol, [51Cr] EDTA and [3H] and [14C]sucrose. 3. In four sheep, permeability measurements for several solutes were made and the results were analysed in terms of restricted diffusion via cylindrical, water-filled pores. Calculation of pore radius was made by a minimum variance method and values ranging from 0-43 to 0-45 nm were obtained. 4. Stability and absence of protein binding of probe molecules was investigated by gel permeation on Sephadex columns.

Bidirectional sodium fluxes across the placenta of conscious sheep.

Conscious pregnant sheep in the last 3 wk of gestation were studied 1--3 days after surgery. Fetal plasma sodium concentration was significantly lower than maternal. A mean electrical potential difference (PD) of 34 +/- 4 (SE) mV (n = 24) was recorded between maternal and fetal intravascular catheters, the mother being positive with reference to fetus. Unidirectional fetomaternal (Jf leads to m) and maternofetal (Jm leads to f) sodium fluxes were determined by application of Fick's principle to uterine and umbilical circulations following injection of 22NaCl or 24NaCl to fetus or mother, respectively. Blood flows were measured by an antipyrine technique. Jm leads to f = 0.142 +/- 0.029 mmol/min (n = 10); Jf leads to m =0.137 +/- 0.015 mmol/min (n = 21). Jm leads to f increased as a linear function of calculated fetal weight. In seven sheep both Jm leads to f and Jf leads to m were measured in a single experiment. The measured ratio Jm leads to f/Jf leads to m was significantly different from the ratio predicted using Ussing's flux ratio equation. There is probably a transplacental sodium pump active in the direction fetus to mother.

Fetomaternal transfer of glucose analogues by sheep placenta.

The steady-state permeability of the placenta to radiolabeled 3-O-methyl--D-glucopyranose (3MeG), alpha-methyl-D-glucopyranoside (AMG), and D-xylose was measured in chronically catheterized conscious sheep near term. Fetomaternal flux was calculated by application of Fick's principle to the uterine circulation after injection to the fetus. At a fetal glucose concentration of 10.2 +/- 1.25 mM, the permeability of 3MeG was half the maximal value of 59.5 +/- 11.2 ml/min found by extrapolation to zero glucose concentration. The permeability to 3MeG is considerably greater than is compatible with restricted diffusion, which together with the competitive effect of D-glucose suggests a carrier-mediated transfer mechanism. The permeability to AMG was less than 0.1 ml/min. The specificity pattern of sugar transport from the fetal side of the sheep placenta is different from that reported for gut lumen or kidney tubule in other species. D-Xylose is not metabolically inert after its injection into the fetal lamb.

Permeability of the human placenta in vivo to four non-metabolized hydrophilic molecules.

1. Permeability of the human placenta to four permeants of different molecular size was measured at Caesarean section in seven normal full-term pregnancies. 2. Placental clearance for mannitol was 8.7 +/- 1.1 ml min-1 (mean +/- S.E.M.), lactulose 6.3 +/- 0.8, chromium ethylenediaminetetraacetic acid (CrEDTA) 3.7 +/- 0.5, and inulin 0.98 +/- 0.1. 3. The permeability data were analysed in terms of restricted diffusion through 'notional' water-filled pores and found to be incompatible with a single population of uniform pores.

Prenatal diagnosis of 3-hydroxy-3-methylglutaric aciduria by GC-MS and enzymology on cultured amniocytes and chorionic villi.

This paper reports the prenatal diagnosis of HMG CoA lyase deficiency at 16 weeks' gestation by direct chemical analysis of cell-free amniotic fluid and by measurement of HMG CoA lyase activity in cultured amniocytes. Termination of an affected fetus allowed study of chorionic villus tissue, the results providing the basis for future first trimester prenatal diagnoses of this condition. An abstract report of this work has appeared elsewhere.

Placental permeability in the sheep.

The transplacental flux of different radiolabelled substances was measured, according to Fick's principle, in chronically catheterised pregnant sheep (124 to 143 days of gestation). Knowing the uterine blood flow, it was possible to calculate the net flux and derive a permeability coefficient, K. With 14C erythritol, K soon reaches its ultimate value, thus suggesting there is only one rate-limiting step to transfer and no significant middle compartment in the sheep placenta. The comparison of feto-maternal fluxes of different metabolically inert, lipid insoluble molecules (urea, erythritol, mannitol and Cr-EDTA) demonstrates a very sharp decrease in permeability as molecular size increases. The membrane behaves like a tight epithelium, with pores of of an approximate radius of 0.45 nm which allow but a poor passive diffusion. On the contrary, permeability to 3 methylglucose and 2 deoxyglucose is much too high for passive diffusion. Transfer competition between D-glucose and 3 methylglucose affords other evidence for a stereospecific monosaccharide carrier. The relationship between the presumably "active" sodium flux out of the fetus and the electrical potential difference across the placenta is discussed. The fetus is usually negative to the mother but the potential difference changes from day to day, and in twin gestation, the respective fetomaternal potentials seem to be independent. Acid infusion to the fetus increases the potential difference. The maintenance of this electrical or chemical gradients is taken as a consequence of the low passive permeability of the placenta.

Radioiodide transfer across sheep placenta.

Unidirectional transplacental clearances of radioiodide were calculated from the net radioiodide fluxes after injection into fetal and/or maternal circulations of 33 catheterized conscious sheep. Maternofetal potential difference (PD) was also recorded. Clearance reached a steady state 20 min after bolus injection. Fetomaternal clearance was related to PD. Bidirectional clearance ratios measured in five experiments showed a significant divergence from the value for passive flux predicted from the measured PD, and in four experiments these ratios were also significantly different from unity, this result being incompatible with passive flux even if the transplacental PD is assumed to be zero. Injection of thiocyanate or iodide reduced radioiodide clearance. Fetomaternal clearance of radioiodide was halved by an increase in fetal plasma iodide concentration of approximately 0.1 mM. There appears to be an inhibitable iodide-transporting site capable of active transport in either direction.