Stephen A. Landaw

Bupivacaine alters red blood cell properties: a possible explanation for neonatal jaundice associated with maternal anesthesia.

Cord blood was incubated with lidocaine, mepivacaine, bupivacaine, or buffer and red blood cell filterability was determined. Only bupivacaine at either 1 or 2 micrograms/ml prolonged filterability by an average of 58 to 65% over red cells treated with buffer alone. Tritiated bupivacaine was bound to a greater extent to red cell ghosts from cord blood (24.6 +/- 5.8%) than to adult red cell ghosts (14.6 +/- 2.6%). Finally, we determined red cell survival in 13-day-old rats injected with bupivacaine or buffer. At 2 h after injection, buffer-treated animals had a red cell survival of 96.9 +/- 3.3%, whereas 2-h survival was reduced to 82.6 +/- 8.7% for the animals injected with bupivacaine. Our results suggest that the neonatal jaundice associated with maternal anesthesia, especially bupivacaine, may be related to the observations that these agents cross the placenta, bind to the red cell membrane and reduce its filterability, resulting in shortened red cell survival.

THE EFFECT OF VITAMIN E ON RED CELL HEMOLYSIS AND BILIRUBINEMIA

The life span of the term infant’s erythrocytes is approximately two-thirds that of the erythrocytes of normal adults.’ The red cells of premature infants have an even shorter life span than do the red cells of term infants.’ The mechanisms responsible for this accelerated senescence are unknown. Premature infants, at birth, are deficient in vitamin E. Deficiency of vitamin E, a naturally occurring antiperoxidant, can produce a shortening of red cell life span.3 The purpose of this investigation was to determine if the presence of vitamin E deficiency in the premature infant during the first week of life played a contributory role in the shortened red cell survival. In addition, since hyperbilirubinemia is, in part, secondary to red cell breakdown, we examined the influence of early vitamin E supplementation on bilirubinemia in preterm infants.

922 THE INHIBITORY EFFECT OF TIN PROTOPORPHYRIN (SN-P) ON HEME DEGRADATION

Sn-P is a potent competitive inhibitor of heme oxygenase, the enzyme which converts heme to bile pigment and carbon monoxide (CO), and has been used to suppress hyperbilirubinemia in experimental animals and man. These experiments were performed to determine which heme pools are affected by Sn-P. Hepatic heme(s) were labeled with delta-aminolevulinic acid (ALA)-5-14C, which does not label hemoglobin heme in the rat. The “free” heme pool was tested by injecting heme prepared in vivo from glycine-2-14C Adult (300g) rats were injected either with Sn-P (50uM/kg sq) or saline one hour before injection with either 10uCi ALA-5-14C or 0.9mg hemin (specific activity 3×104 DPM/mg in 1ml alkalinized rat plasma). Expired 14CO was then collected over the next 24 hours. Results were:These experiments indicate that a single injection of Sn-P significantly inhibits the degradation of hepatic (non-erythropoietic) hemes for 12 hours and exogenous heme for an even longer period. They further suggest that Sn-P may exert different degrees of inhibition of bilirubin (and CO) formation depending upon the heme pool(s) involved.

192 DECREASED SURVIVAL AND ALTERED MEMBRANE PROPERTIES OF RED BLOOD CELLS (RBC) IN THE NEWBORN RAT

Decreased survival has been noted for RBC of newborn man and rat(Ped.Res.11:1155,1977).The present studies investigate the possibility that decreased survival is due to an altered RBC membrane. Osmotic fragility of newborn(NB) rat RBC was increased (29% lysis in 0.5% NaCl;adult RBC:6%),increasing to 92% following 1 hr incubation at 20° in pH 9.2 borate buffer (adult RBC: no change). Filtration half-time of RBC suspensions was prolonged (NB:40 min; adult:2-3 min)and returned to normal with advancing age(16 min at 6 days,5 min at 12 days,3 min at 26 days). Splenic uptake at 24 hr was increased 15-fold & hepatic uptake increased 3-fold for 51Cr-labeled NB RBC as compared to adult RBC. Following treatment with the cross-linking agent dimethyladipimidate (DMA,2-5mM,pH 9.2),survival of 51Cr-labeled adult RBC was slightly reduced from 100&92% at 1&24 hr to 94&76%. DMA(2-5mM) caused marked further reduction in survival of NB RBC from 64&16% at 1&24 hr to 10&3%. Following incubation of intact RBC with glucose, electrophoresis of RBC membrane proteins yielded a pattern of high molecular weight complexes(HMWC) for NB,but not for adult, RBC. These results suggest an unstable RBC membrane in the newborn rat,leading to altered osmotic fragility,reduced deformability, splenic trapping, and reduced survival. Preferential hemolysis of DMA-treated NB rat RBC and HMWC formation suggest that an altered state of aggregation of membrane proteins may underlie these abnormalities.

1269 POSSIBLE RELATIONSHIP BETWEEN MATERNAL ANESTHESIA AND NEONATAL JAUNDICE: EFFECT IN VITRO OF ANESTHETICS ON THE NEONATAL RED CELL

Neonatal jaundice has been correlated with use of anesthetics in the mother. These membrane-active agents cross the placenta & lead to measurable blood levels in the newborn. We investigated the effect in vitro of 3 commonly used agents on filterability of neonatal red cells (RBC). Cord blood was obtained from 10 infants whose mothers had received no medication or anesthetics prior to or during labor. RBC were washed and resuspended to a hematocrit of 5% in pH 7.4 Krebs phosphate buffer, & incubated for ½ hour at 37°C with either buffer or anesthetic agent (1 or 2 μg/ml). Filterability was tested using a modification of Teitels method.For the 3 agents tested, no significant difference was seen in T1/2 at the 2 concentrations tested. At both 1&2 μg/ml, the T½ of bupivicaine-treated RBC was significantly prolonged when compared with buffer-treated RBC. These results complement available clinical information linking the use of bupivicaine with neonatal jaundice, and suggest that this agent may be adversely affecting the deformability of neonatal RBC, leading to premature red cell destruction.

662 ASPIRIN (ASA) AND HYDROGEN PEROXIDE (H 2 O 2 ) INDUCED RED BLOOD CELL INJURY, MEMBRANE PROTEIN ALTERATIONS

Previous studies have demonstrated that normal intact red blood cells (RBCs) will undergo membrane lipid peroxidation and will hemolyze in vitro in the presence of ASA+H2O2 but not with either agent alone (Ped. Res. 9:326, 1975). In order to determine whether ASA+H2O2 induced oxidant injury results in RBC membrane protein damage, 5% RBC suspensions were incubated with equal volumes of buffer, ASA (25 mg%), H2O2 (1.2%) or ASA (25 mg%)+ H2O2 (1.2%). Following incubation, RBC membrane proteins were separated on SDS gel. ASA+H2O2 resulted in the production of a high M. Wgt. membrane protein complex (M. Wgt. 300,000-600,000) with a decrease in other membrane protein fractions. Similar changes were not observed with ASA alone or H2O2 alone. Incubation with 14C-acetyl and 14C-carboxyl tagged ASA+H2O2 failed to label any membrane protein fraction. Although prior incubation with water soluble ≥ tocopherol in concentrations as low as 0.01 mg/ml completely prevented hemolysis and membrane lipid peroxidation in the presence of ASA+H2O2, no protective effect on RBC membrane protein was found. While in this system lipid peroxidation is more predictive of in vitro hemolysis than is the membrane protein change, such protein change, if it were to occur in vivo, might result in hemolysis. ASA+H2O2 induced hemolysis represents an interesting model by which to study the multiple effects of oxidative damage on the normal intact RBC and the mechanism of the protection afforded by antioxidants.

IRON ABSORPTION FROM HUMAN MILK, SIMULATED HUMAN MILK AND PROPRIETARY FORMULAS

Studies from our laboratory have shown that iron is better absorbed fron human milk than from cow milk and can provide sufficient iron for infants during their first year. This study was designed to compare iron availability from human milk with other formulas and determine factors responsible for its superiority. Adults were fed 100 ml of the following: human milk (HM), simulated human milk (SHM), simulated human milk containing added lactoferrin (SHM-L) and 2 commercial formulas (CF) containing iron, 12 mg/qt. The SHM resembled HM in concentration of protein, fat, carbohydrate, iron, total minerals, calcium and phosphorus. Fe59 was added to each feeding and iron incorporation into red cells was determined 14 days after each feed. Iron absorption was highest from HM (15.7%) and lowest from one of the CF (1.7%). The SHM supported a 9.3% absorption; addition of lactoferrin reduced this to 4.7%. Net iron absorption was 0.12 mg/L from HM and 0.20 and 0.37 mg/L from the iron enriched CFs. This study demonstrates that the enhanced iron absorption from HM is not a simple result of its gross composition or the presence of lactoferrin, and that HM, with only 6% of the iron of a leading CF, can provide as much as 60% of the iron derived from such a proprietary milk.

Lack of relationship of red cell enzyme activity to bilirubin and carboxyhemoglobin levels in healthy term infants.

ABSTRACT: Komazawa, M., Landaw, S. A. and Oski, F. A. (Departments of Pediatrics and Medicine, State University of New York, Upstate Medical Center, Syracuse, New York, USA). Lack of relationship of red cell enzyme activity to bilirubin and carboxyhemoglobin levels in health term infants. Acta Paediatr Scand 64:473, 1975.–A total of 32 term infants were studied in an attempt to confirm and extend the recent observation of Petrich & associates (14) that minor degrees of transient deficiencies of the red cell enzymes glucose phosphate isomerase and glyceraldehyde‐3‐phosphate dehydrogenase were related to hyperbilirubinemia in otherwise healthy term infants. No relationships could be observed between the activity of these enzymes and the bilirubin level on day three. In addition, no correlation was present between bilirubin values and carboxyhemoglobin levels on day three, suggesting that in the healthy term infant excessive hemolysis is not usually responsible for the variations observed in bilirubin levels.