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Cytotrophoblasts isolated from normal human placenta cultured under normoxic conditions (20% O2, pO2 = 130 mmHg) for 48-72 h differentiate to a form which expresses high levels of hCG and which morphologically resembles syncytiotrophoblast. We had previously shown that hypoxia (0-1% O2, pO2 = 12-14 mmHg) blocks this differentiation process, although trophoblasts exposed to hypoxia for up to 96 h were completely viable. In this article we showed that trophoblast responds to hypoxia by expressing the hypoxia-sensitive DNA binding protein HIF-1. We also showed that in trophoblast cultured under normoxic conditions, expression of endothelial cell nitric oxide synthase (ecNOS) mRNA increases with time, reaching a maximum in 48-72 h. However, in trophoblast maintained under hypoxic conditions for 48 h (after an initial 24 h in normoxia), expression of ecNOS mRNA is greatly reduced. These observations are consistent with the expression of ecNOS by syncytiotrophoblast but not by cytotrophoblast. In contrast, exposure of differentiated trophoblasts to hypoxia for 24 h (after 48-72 h in normoxia) significantly stimulates expression of ecNoS mRNA over that of cells maintained continuously in normoxia. These results suggest that in differentiated trophoblast hypoxia can stimulate ecNOS expression.

The mechanism of IgG transport by the placental trophoblast was examined by studying IgG uptake by purified trophoblast maintained in culture. This model retains the ability to bind and endocytose human IgG from human serum. Comparison of the relative IgG uptake by the trophoblast among the four subclasses of both human and mouse IgG indicates that the trophoblast IgG receptor has different affinities from those described for the three known human Fc gamma receptors, FcR gamma I, FcR gamma II, and FcR gamma III. These results suggest the presence of a novel trophoblast Fc gamma receptor. Although Fc gamma RIII has been reported to be present on trophoblasts, immunocytochemical studies failed to detect binding to the cell surface of antibody-specific for Fc gamma RIII, 3G8 MAb. In addition, blocking studies with MAb 3G8 did not interfere with IgG uptake. Scatchard analysis of human IgG uptake revealed a biphasic curve consistent with two distinct mechanisms for the transport of IgG by the trophoblast. The first is a higher affinity system (Ka = 1.7 x 10(7) M-1, 1.7 x 10(4) binding sites/cell) which exhibits IgG subclass and species specificity, and the second is a low affinity system (Ka = 6.9 x 10(3) M-1, 7.5 x 10(7) binding sites/cell).

Trophoblast from term and first trimester placenta, maintained in culture, were exposed to 20 μmoles/1 zidovudine or 2′3'dideoxyinosine. Several indices of function were measured and compared to control trophoblast in parallel culture. The results from individual placentas were examined by Student's t-test and cumulative results by ANOVA. Neither zidovudine or 2′3'-dideoxyinosine had statistically significant effects on the function of term trophoblast, following a 48 hr exposure to the drug as indicated by hCG secretion, protein synthesis and glucose consumption. In one of five placentas exposed to zidovudine, progesterone secretion was reduced as compared to its control but remained in the high range. Zidovudine had no significant effect on cultured trophoblast isolated from first trimester placenta even after prolonged exposure to the drug for eleven days. Both term and first trimester trophoblast in culture tolerate prolonged exposure to high concentrations of zidovudine or 2′3'-dideoxyinosine. Human trophoblast in culture provides a safe in vitro model for the screening of drugs intended for use during pregnancy.

Our purpose was to investigate the transfer of cocaine across human placenta and to measure the binding of cocaine to maternal and cord sera and to assess the effect of binding on transfer.Cocaine transfer by the in vitro perfused human placenta was studied under controlled experimental conditions. Protein binding of cocaine was measured by ultrafiltration in 10 pairs of maternal and cord sera and was compared with 12 sera from nonpregnant females.With perfusates of albumin (5 gm/dl) in buffer cocaine clearance was 1.08 +/- 0.52 ml/min, threefold greater than that of the water-soluble marker L-glucose. Transfer was bidirectional and nonsaturable over a concentration of 0.02 to 4000 ng/ml. Cocaine was not detectably metabolized during perfusion. Replacement of albumin-buffer with human serum as maternal perfusate reduced the transfer rate by almost 50%, p0.02. Binding of cocaine was greatest by serum from the nonpregnant femalepregnant female (not significant)cord serum (p0.02) = albumin buffer.Cocaine is rapidly transferred across the placenta by simple diffusion without metabolic conversion. Transfer, although diminished, remains rapid in spite of binding to serum proteins. These several factors plus the poor binding by cord serum conspire to increase fetal exposure to the drug.

The transfer of zidovudine (AZT) across human placenta was studied using an in vitro perfusion system with independent maternal and fetal circulations. AZT is transferred toward the fetus more rapidly than L-glucose (transfer index 1.5), a water-soluble molecule smaller than AZT (267 vs. 180 Da) that passively diffuses across the placenta. The transfer rate is proportional to the concentration in the maternal perfusate over a range of 0.03-300 microM. Transfer rate in the reverse direction, toward the maternal perfusate, also exceeds that of L-glucose and fails to show saturability. These observations are consistent with simple diffusion. The partition of AZT and glucose between perfusion buffer and octanol is 1.04 and 0.013, respectively, indicating that AZT is more lipophilic and providing a reasonable explanation for the more rapid transfer. AZT is extensively metabolized by the placenta to more polar and as yet unidentified metabolites that are not released into the perfusate. The possibility that the placental accumulation of such metabolite(s) may exert an antiviral action and may also affect placental function must be considered.

OBJECTIVE: Our purpose was to determine whether human trophoblast has a cell surface CD4 antigen that will bind to gp120, the envelope protein of human immunodeficiency virus. STUDY DESIGN: Uptake of iodine 125 - labeled gp120 by trophoblast in culture was measured. Particular attention was paid to technical details that may have caused the contradictory results reported by previous investigators: the source of the recombinant gp120, the method of radioiodination, and the isolation procedure of trophoblast to ensure elimination of contaminating cells, particularly macrophages. RESULTS: Uptake of transferrin-free iodine 125 - labeled gp120 to trophoblast was unaffected by adding a 200 molar excess of gp120, by preincubating gp120 with soluble CD4 to block the CD4 binding sites on gp120 and by preincubation of trophoblast with a blocking antibody to CD4 (OKT4a). In contrast, uptake of gp120 by CD4-positive H9 human lymphocytes was reduced 79% by a 200 molar excess of gp120 and >50% by a CD4-blocking antibody. CONCLUSIONS: Uptake of gp120 to trophoblast is by a high capacity, CD4-independent mechanism that is probably nonspecific and may be related to the mechanism for binding other circulating glycoproteins in maternal blood. (AM J OBSTET GYNECOL 1996;174:49-54.)

The transfer and metabolism of retinol by human placenta was investigated using an in vitro perfusion system with independent maternal and fetal circulations. 3H-retinol bound to albumin added to the maternal perfusate was rapidly taken up and concentrated by the placenta to levels 16.5 ± 5.28 times the maternal perfusate. Approximately 8% of the retinol retained in the placenta was esterified. No metabolites were detected in the perfusates. Perfusion of placenta with retinol bound to retinol-binding protein (RBP) reduced the placental concentration to 4.4 ± 1.72 times the maternal concentration and eliminated evidence of metabolism. The transfer rate of RBP:3H-retinol was less than that of albumin:14C-retinol when measured concurrently in three experiments (clearances 0.11 versus 0.75 mL/min, 0.21 versus 1.7 mL/min, and 0.29 versus 0.48 mL/min, respectively). Transfer of the radioactive retinol was more rapid than 125I-RBP or albumin, indicating that retinol was transferred independently of the proteins. The transfer index of retinol (clearance reti-nolxlearance L-glucose) was 0.73 ± 0.085 compared to 2.1 ± 0.36 for thiamin and 3.4 ± 0.95 for riboflavin, both water-soluble vitamins with active transport systems. The retinol transferred to the fetal perfusate is not bound to RBP, as demonstrated by gel filtration chromatography and chromatography on a transthyretin affinity column, despite the availability of RBP in the cord serum added to the perfusate. The endogenous retinol in the cord serum is bound to RBP. The conclusions derived are that RBP binding reduces the accumulation of retinol in the placenta and the transfer to the fetus, that retinol is transferred intact to the fetal circulation where immediate binding is not to RBP, and that redistribution to RBP occurs subsequently, possibly in fetal liver.

In this discussion of in vitro perfusion of human placenta as an approach to the study of fetal nutrition I shall: (1) explain why, in recent years, we have discontinued using experimental animals and concentrated on human placenta; (2) describe the perfusion technique; (3) review our studies of two nutrients, glutamic acid and riboflavin, to illustrate the flexibility of experimental design that is possible and the type of information that can be obtained; (4) summarize the limitations and the potential of the perfusion technique as we have observed them.

RESPONSE OF HUMAN TROPHOBLAST TO HYPOXIA: MORPHOLOGY, GLUCOSE TRANSPORT AND METABOLISM, ENDOCRINE SYNTHESIS. † 1230

One of Saul Krugman's favorite photographs recalls the day in 1965 when he handed his predecessor at New York University, L. Emmett Holt, Jr., a copy of the Festschrift in his honor. Some 25 years later, it is our privilege to similarly honor Saul. It continues a long tradition at Bellevue Hospital. A Festschrift was dedicated to Abraham Jacobi, the first Director of Pediatrics, some 100 years ago. Tradition plays an important role in Saul Krugman's life and in the history of medicine in New York City. New York hospitals have long been involved with the sequestration and care of patients with infectious diseases. In fact, many municipal hospitals were established because of epidemics of smallpox, measles, typhus, yellow fever, cholera, and typhoid fever. To protect the general population from infections arriving on ships, quarantine sites were established on islands in New York Harbor and the East River. These quarantine hospitals became the pest houses. The word "pest" derives from the first of the three great scourges of mankind: pestilence, famine, and war. As New York's municipal hospital system became better established, the pest houses evolved into infectious disease hospitals. The most famous of these was Willard Parker Hospital located on the East River on Manhattan's lower East side. Saul was discharged from the Air Force in 1946 and joined an army of veterans seeking hospital training. Because of a delay in procuring a Pediatric residency at NYU-Bellevue, he took an interim position as resident physician at Willard Parker for six months. At the time all patients with infectious diseases in New York were funnelled into three specialty hospitals. Waves of children arrived at Willard Parker with scarlet fever, measles, pertussis, croup, and polio, filling wards to overflowing. Saul became an expert in infectious disease even before he began his pediatric residency! He maintained his affiliation with Willard Parker until it was closed 10 years later, signaling the end of an epoch as the result of medical advances. During those momentous years, Saul sharpened his clinical skills to an extraordinary degree, laying the groundwork for his future career in research and accumulating the experience that made the Krugman and Ward text, Infectious Diseases of Children, now in its 9th edition, an international success. The past 100 years has seen the conquest in this country of most of the dreaded infections that concerned us at the opening of the century. Saul Krugman participated in many of those advances. It is part of the irony of medical progress that, as we congratulate ourselves on our success, a new threat has emerged which will tax our energies and resources for many years to come. The frightening dimensions of AIDS will stalk the 21st century.

The workshop on in vitro perfusion of human placenta concentrated on recent advances that might influence the use of the techniques. Presentations were made by Henning Schneider, Nicholas Illsley, and Richard Miller. Maurice Panigel and Heinz-Peter Leichtweiss introduced the discussion The allotted time passed quickly, with questions and conversations overflowing into the corridors during the rest of the conference. In the following, we shall review briefly the workshop and attempt to summarize the ensuing conversations in a form that we hope will be useful to the reader.

Summary Transfer of amino acids was studied with an in vitro perfusion technique of human placenta in which independant maternal and fetal circulations were established. When the fetal perfusate was recirculated, a concentration gradient was established with fetal concentrations of alanine, glycine and lysine about 1.5 times higher than maternal. In this system, the placenta could actively transfer 4.1 mM per day of each of these amino acids. Glutamic acid and aspartic acid were progressively removed from the fetal circulation so that fetal concentrations fell below maternal. The exchange rates of L- and D-glutamic acid were compared, the latter providing a measure of simple diffusion. The ratio of L:D exchange rates was 0.64 from the maternal to the fetal circulation and 0.41 in the reverse direction, indicating a more rapid bidirectional exchange of the D-isomer. Simultaneously measured exchange rates of leucine gave a L:D ratio of 1.73 and 1 00 respectively, indicating active transfer of L-leucine towards the fetus. While for L-glutamic acid, transfer across the placenta was even lower than expected on the basis of simple diffusion, uptake into the tissue was high. Of the amount presented to the placenta, 49 percent was taken up, of which only 10 per cent passed to the other side. The rest was retained and presumably metabolized. For L-leucine, the uptake was only 16 percent of which 75 per cent was transferred. The placenta serves as an effective barrier to the transfer of glutamic acid by rapidly metabolizing this amino acid. It is postulated that maternal glutamine, which is efficiently transferred across the placenta, contributes its amid-N2 to the fetal synthesis of purines and pyrimidines and that the resulting surplus of glutamic acid is returned to the placenta which removes it from the circulation and metabolizes it.

The metabolism of D- and L-pipecolic acid has been investigated in rabbits and rats. A rapid evolution of 14CO2 followed the injection of either D- or L-pipe[6-14C]colic acid into rabbits. Rabbit kidney slices degraded to CO2 both isomers of lysine and of pipecolic acid. Rabbit liver was effective with only the L-isomers. In the rat, very little of injected L-pipecolic acid was catabolized to CO2, and large amounts were excreted unchanged into the urine, L-Lysine was efficiently metabolized to CO2 by rat liver and kidney slices but not D-lysine or either isomer of pipecolic acid. Rat kidney converted D-lysine to L-pipecolic acid. The L-isomer was identified by co-precipitation of the radioactive product with authentic compounds.

The transfer of radioactive riboflavin across the human term placenta has been studied in an in vitro perfusion system. The clearance index (clearance riboflavin:clearance antipyrine) toward the fetus averaged 0.69 and the transfer index (clearance riboflavin:clearance Lglucose) averaged 3.40. The respective indices in the reverse direction were 0.25 and 0.87. Stepwise increases in the concentration of riboflavin in the maternal perfusate were associated with parallel increases in transfer rates, expressed as ng/min, up to concentrations approximating 100 ng/ml. Above that concentration, the transfer rates continued to increase at a slower rate. Concurrently, there was a reduction of the transfer index from 2.7 to 1.1 at 1000 ng/ml. With the fetal circulation closed, the placenta established a gradient toward the fetus over a period of 150 min of 1.7. The transferred radioactivity was identified as riboflavin by high-performance liquid chromatography, whereas that retained within the placenta was metabolized to flavin mononucleotide (33-75%). The observations indicate a very effective active transport system directed toward the fetus which is limited in capacity to low concentrations of riboflavin.

Extract: Human placentas were perfused in vitro with straight chain, even-numbered free fatty acids (FFA) and the transfer rates from maternal to fetal circulation were measured. The transfer rates increased logarithmically as chain length decreased. The clearances, calculated as percentage of clearance of antipyrine were: palmitic acid, 4%; myristic acid, 10%; lauric acid, 16%; decanoic acid, 18%; octanoic acid, 50%; hexanoic acid, 64%. Speculation: Transfer of hydrophobic materials through the placenta is a complex process which is probably affected by many factors. The present studies with FFA suggest that, for these compounds, protein binding may be particularly significant. If so, it may affect transfer rate by influencing release from maternal serum albumin, diffusion through the placenta, and uptake by fetal plasma. These several factors require further analysis.

Extract: The effect of protein binding on the rate of placental transfer of hexanoic (C 6) and decanoic (C 10) acids was investigated in an in vitro perfusion system of human placenta. As much as 30% of transferred C 6 was converted to more polar compounds; so that the observations related to the combined effects on transfer and metabolism. Less than 10% of C 10 was similarly metabolized. Both fatty acids are soluble in buffered salt solutions at the concentrations used (40 μM) and both are bound to serum albumin, C-10 having higher association constants (K′ for C 6, 1.48 × 104; for C 10, 1.03 × 105). When the placenta is perfused with buffered salt solution, the transfer of C 6 is 22% more rapid than that of C 10. It is suggested that binding within the placenta retards C 10 more than C 6. The addition of 1 g/100 ml bovine serum albumin to the maternal perfusate reduces the transfer rate of C 10 by 80%, whereas 2 g/100 ml serum albumin has a more moderate effect on C 6 (a reduction of 50%). The addition of 1 g/100 ml serum albumin to the fetal perfusate increases transfer rate of both free fatty acids (FFA), C 6 by about 25% and C 10 by about 250%. With equivalent concentrations of serum albumin in maternal and fetal perfusates, the transfer rate of C 10 was reduced by 65%, whereas there was no detectable effect on transfer of C 6 in two of three experiments. The transfer rates of FFA increase logarithmically with progressive shortening of the carbon chain from C 16 to C 8 when maternal and fetal perfusates contain serum albumin. Protein binding is apparently the determining factor. The rate of transfer falls off at C 6 and C 4, even though protein-binding continues to decrease. The determining factor may be the hydrophilic nature of these molecules. Speculation: If the observations on FFA have general applicability, the overall effect of protein binding on placental transfer of a particular substance may relate to its water solubility and protein binding. If water solubility is not a limiting factor, protein binding may decrease transfer rate sharply, as occurs with decanoic acid (C 10). With substances that have very limited water solubility, protein binding may increase transfer by increasing the amount presented to the placenta. Palmitic acid (C 16) appears to be such a compound. The fraction bound to proteins in the perfusate, as measured by equilibrium dialysis, may not be the most significant factor determining rates of transfer. More significant may be the competitive aspects of binding in the perfusate and to the placenta in influencing rates of release and uptake.

The kinetics of riboflavin transfer across the perfused human placenta has been studied. With both circulations open, the transfer index (clearance of riboflavin: clearance of antipyrine) toward the fetus was 3.3 +/- 0.08 (+/- SD) versus 0.87 +/- 0.02 in the reverse direction (p less than 0.01). Galactoflavin reduced the materno-fetal transfer rate. The uptake of riboflavin and L-glucose after bolus injection into the maternal perfusate was 35% +/- 9.6% versus 3% +/- 16%, p = 0.005, which indicates an uptake rate in excess of diffusion. There was no significant difference in uptake at the fetal surface. With recirculation of either the maternal or fetal perfusate, the transplacental gradient that was established was lower at high concentrations of riboflavin, which indicates the saturability of this function. Riboflavin was concentrated in the intracellular space and partly metabolized to flavin mononucleotide and flavin adenine dinucleotide. An equilibrium was approximated when both perfusates were recirculated, with the concentration lowest in the maternal perfusate, highest in the placenta, and intermediate in the fetal perfusate. It is concluded that the major factor directing riboflavin toward the fetus is the difference in rates of uptake at the two surfaces of the placenta.

Zellweger syndrome is the prototype of a growing group of genetic diseases caused by an absence or deficiency of peroxisomes. The defect causes the enzyme catalase to remain in the cytosol instead of being packaged into peroxisomes. This mislocalization can be easily detected by sedimentation analysis. Amniocytes were homogenized and then centrifuged to pellet organelles. Catalase was found to sediment with the peroxisomes in the homogenates of normal cells, but to remain in the supernatant with Zellweger syndrome amniocyte homogenates. This striking difference is unambiguous and reproducible, and provides a simple method for prenatal diagnosis. Moreover, it allows one to differentiate diseases in which peroxisomes are deficient from other peroxisomal diseases in which the organelle is intact, but one enzyme is defective. Electron microscopic observations support the biochemical determinations. Normal amniocytes contain small peroxisomes in which a weak cytochemical reaction for catalase may be demonstrated. Zellweger amniocytes appear to lack these organelles, although some cells have rare structures that might be residual or abnormal peroxisomes.

In familial dysautonomia there are malfunctions of motor, sensory and autonomic systems. The sural nerve has reduced transverse fascicular area, diminished numbers of myelinated axons (particularly those of small diameter) and very few non-myelinated axons. Catecholamine containing endings are not found in accompanying arteries. These changes are compatible with reduced neuronal populations described in sympathetic and sensory ganglia. The observed pathology accounts for many of the clinical features of the disease and suggests an abnormality in intrauterine development.

Purified rat peroxisomes have been reported to oxidize D -pipecolic acid and the pipecolaturia of Zellweger syndrome has been attributed to the absence of peroxisomes. The logical consequences would be excesses of D -pipecolic acid in the urine of patients and Zellweger syndrome. The urine of two patients with Zellweger syndrome has been analyzed by complexing the pipecolic acid to copper-aspartame to separate the L - and D -isomers. L -Pipecolic acid constituted 100% and 78% of the total pipecolic acid in the two urines. The possibility of preferential retention of D -pipecolic acid was excluded by measuring renal excretion in two control subjects following administration of each isomer. The clearance of L -pipecolic acid was 1.1 and 0.2 ml/min and of D -pipecolic acid was 36.4 and 43.6 ml/min. These results do not support the contention that the pipecolaturia of Zellweger syndrome is the direct result of peroxisomal deficiencies.

Electrophoretic studies of human fetal plasma have demonstrated a protein pattern in which all major fractions are present (1). The site of origin of these plasma proteins is not known. Presumably they may be synthesized by the mother and merely circulate in the fetus as the result of placental transfer, or they may be synthesized by the placenta or the fetus. The gamma globulin level is high at birth and falls during the first weeks of life (2). This led to the assumption that the gamma globulin found at

The ability of human tissues to convert lysine and α-ketoglutarate to saccharopine [ e-N-( l -glutaryl -2)- l -lysine ] has been investigated vitro. Liver is the most effective organ, though some activity could be demonstrated in several other tissues. The responsible enzyme lysine-ketoglutarate reductase (lysine:α-ketoglutarate: TPNH oxidoreductase ( e-N-[ glutaryl -2]- l -lysine forming )) has been partially purified from human liver and optimal conditions for its assay determined. There is a specific requirement for TPNH that cannot be satisfied by DPNH. A pH optimum near neutrality and inhibition by ammonium sulfate were observed. The enzyme is stable at −25°, both in tissues obtained at post-mortem and in its purified form. The assay for lysine-ketoglutarate reductase is sufficiently sensitive to be used on the limited amount of tissue obtainable by needle biopsy of the liver.

Radioautographic examination of the bone marrow of three patients with X-linked congenital hyperuricosuria (Lesch-Nyhan's disease), after incubation with radioactive hypoxanthine, revealed no labeling even in extremely immature blood cells. An adequacy of the cosubstrate, phosphoribosepyrophosphate, in these cells was demonstrated by the appearance of labeling following incubation with tritiated adenine. It is concluded that the enzyme IMP pyrophosphorylase (inosinate: pyrophosphate phosphoribosyltransferase E.C.2.4.2.8) is deficient in young blood cells as well as in peripheral blood elements. The possibility that the enzyme deficiency in the peripheral blood results from the rapid degradation of an unstable enzyme is thereby excluded. Absence of radioactive labeling of terminal cells—erythrocytes, granulocytes, and platelets—is attributed to an insufficiency of the cosubstrate, phosphoribosepyrophosphate.

The available data indicating the optimal conditions for premature infants are reviewed. The deficiencies in our knowledge are stressed. The designs of incubators now available are discussed and their effectiveness in controlling environmental conditions is outlined. The necessity of frequent checking and servicing of the incubators to insure proper performance is emphasized.

Adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) has been purified 4000-fold from human erythrocytes. On Sephadex column chromatography or polyacrylamide gel electrophoresis multiple peaks were observed. The prior addition of 5-phosphoribosyl-1-pyrophosphate (P-Rib-P-p) to the enzyme produced a single peak with a molecular weight of 26 000. Magnesium is not required to form the P-Rib-P-P adenine phosphoribosyltransferase complex. The complex is more stable to heat denaturation. Rabbit antiserum against human adenine phosphoribosyltransferase cross-reacts with lysate of erythrocytes of rhesus monkey but not with those of rabbit, dog, mouse and sheep. Neutralization of rhesus adenine phosphoribosyltransferase is more effective than of human enzyme indicating structural differences between the enzymes. The antiserum neutralizes the same amount of adenine phosphoribosyltransferase activity in erythrocytes from Lesch-Nyhan patients and normal subjects. The enzymes from both sources are not distinguishable by radial diffusion against antiserum or by immunoelectrophoresis. This evidence of immunological identity is consistent with the postulate that the elevated levels of adenine phosphoribosyltransferase found in Lesch-Nyhan patients are due to stabilization of the enzyme by endogenous metabolites.

The metabolism of estrone-16-C14 was studied in 3 human fetuses of 3 to 5 months gestation. About one-third was converted to water-soluble metabolites. By enzymatic hydrolysis and paper chromatography these were shown to be virtually all estrogen sulfates. A small amount of the estrone was converted to estradiol-17β but no estriol was detected. Estrone-16-C14 was sulfurylated efficiently in the 105,000 ×g supernatant fraction of guinea pig placenta, but not in term human placenta.

The transfer of I 131 antipyrine across the guinea pig placenta was measured with the technique of in situ perfusion. The amount transferred per unit volume of perfusate was not affected by alterations in flow rate or intravascular pressure. The amount of radioactivity transferred per unit time was therefore proportional to the flow rate and served as an accurate index of blood flow. The amount of sodium transferred across the placenta from fetus to mother was measured with Na 22 as tracer. The transfer per unit volume varied inversely with the flow rate while transfer per unit time varied directly with flow rate. Radioactive sodium provides a less accurate measure of flow rate than antipyrine, but is a better indicator of the permeability of the placental membrane.

In a recent report from these laboratories (2), an in situ perfusion method was described which could be used to measure the transfer of substances in either direction across the guinea pig placenta. When the procedure was applied to the estrogens, it was found that the unconjugated hormones, estrone, estradiol170, and estriol were transferred quickly under conditions in which the placenta posed an effective barrier to their glucuronide conjugates. However, it was found that when estrogens were administered to the mother, conjugates other than glucosiduronates were detected in the umbilical circulation. In the present report, evidence is presented indicating that these conjugates are estrogen sulfates, that the placenta is impermeable to these conjugates, but that the placenta can sulfurylate estrogens and release them into the umbilical circulation.

The metabolism of the peripheral leucocyte has been studied in a child presenting with mental retardation and hypervalinemia. A defect in the transamination of valine was demonstrated. Evidence is presented that the transamination of value is dependent on an enzyme that is specific for valine. Transamination of valine is demonstrable in placenta, suggesting the possibility that the diagnosis may be made before birth.

Forty infants from 1 week to 6 months of age who had no circulating antitoxin were immunized with one injection of a high titer diphtheria toxoid on alum and their antitoxin responses were followed at regular intervals up to six months afterwards. The ability to form antitoxin improved significantly during the first two months of life. No significant difference could be demonstrated between the response of infants 2 to 4 months of age and that of infants 6 months of age. (In the statistical tests, the 5% level was adopted as indicating significance.) Twenty-seven of these infants were immunized at the same time in the opposite arm with a single dose of tetanus toxoid on alum. The response to tetanus immunization was faster and better in infants from 2 to 6 months of age than in infants 1 month of age and younger. The response to the tetanus toxoid preparation was faster and better than the response to the diphtheria toxoid preparation. The following conclusions are drawn: A. The infant can form antibodies from birth. B. The ability to form antibodies improves during early life. For diphtheria and tetanus antitoxin, the improvement is rapid during the first two months and then is much slower. C. The advisability of early immunization is related to the potency of the antigen to be used.

The effects of Urecholine have been investigated in six children with familial dysautonomia. There was an increase in eye moisture, a reduction in gastric distension and vomiting, improvement in esophageal motility, and better bladder control. Urecholine may prove an effective therapeutic agent for selected patients with familial dysautonomia.

Extract: Maternal rats fed a diet deficient in potassium (K) during pregnancy were depleted of K so that the plasma level of K fell to one-half the normal value and the concentration in maternal muscle fell by about 30%. The K concentration in fetal plama under these circumstances did not change significantly, but that in fetal tissues (placenta and fetus) decreased by roughly 10% (fig. 1, table III). Maternal hyperkalemia, induced by a sodium-deficient diet or produced by infusions of K (table IV) induced a fetal hyperkalemia (fig. 1). Maternal hyponatremia caused proportional hyponatremia in the fetus (slope = 0.60, correlation coefficient = 0.91) (fig. 2); the sodium content of the fetus was also reduced in maternal hyponatremia (fig. 3). Speculation: Deserving of particular emphasis is the contrast between the effect of maternal hyperkalemia and hypokalemia on the level of potassium in the fetal blood. This difference in response raises questions concerning the mechanism of placental transport of potassium. It would be of interest to determine if the placenta protects the fetus against other maternal ion deficiencies.

The response to increased carbon dioxide (4 per cent) and reduced oxygen (12 per cent) inhalations in subjects with dysautonomia has been compared to that in normal individuals. In both instances, the patients with dysautonomia revealed a reduced ability to cope with these stresses, with potentially serious consequences. These observations are significant in the pathophysiology of familial dysautonomia and suggest approaches to therapy.

The enzyme defect in maple syrup urine disease and in its varient form, intermittent branched-chain ketonuria, is demonstrable in skin fibroblast cultures derived from patients with this disease. In both instances there is considerable reduction in the ability to decarboxylate all three branched-chain keto acids. In view of the recent evidence that there are two or three branched-chain decarboxylases, each with a specific substrate, it is suggested that one gene controls the synthesis of a polypeptide that is common to the involved enzymes or to some structural or regulatory part of the enzyme complex.

Four human fetuses of 4–5 months of gestation were perfused with estrone-6,7-3H sulfate-35S. The doubly labeled estrogen was used to follow the metabolism of estrone sulfate and to determine the extent of metabolism occurring without hydrolysis. Radioactivity was found in all the examined organs but was most concentrated in liver and kidney. The radioactive metabolites contained in liver were further investigated in 2 fetuses, using different analytical procedures. In the first instance, there was evidence for several unidentified estrogen sulfates, all with a 35S/3H ratio the same as that of the perfused estrone sulfate, indicating metabolism of the estrone radical without prior hydrolysis. In the second liver extract, estrone sulfate contributed 13%, estradiol sulfate 1.7% and unidentified estrogen sulfates most of the remaining radioactivity. A constant 35S/3H ratio of the various estrogen sulfates at the level of the perfused estrone sulfate again indicated metabolism without prior hydrolysis. Estriol...