Jack Metcoff

Relation of Protein Synthesis to Plasma and Cell Amino Acids in Neonates

ABSTRACT. We hypothesized that more rapidly growing preterm infants would have higher rates of protein synthesis than term infants, and that protein synthesis would be more closely related to intracellular than plasma levels of amino acids. Neutrophils, used as a cell model, were isolated from 1-3 ml blood of 63 infants 27-44 wk postconceptual age. Protein synthesis (3H-leucine incorporation, pmol/h/mg DNA), and 19 amino acids in the leukocytes (nmol/mg DNA) and plasma (nmol/ml) were quantified. Protein synthesis was related inversely to birth weight and gestational age, i.e. the smaller and more preterm the infant the higher the rate of protein synthesis. Multiple regression analysis limited to six steps indicated that some plasma amino acids (Val, Ile, Phe, Asp, Ala, Tau) accounted for a significant (p = 0.03), but relatively small, proportion, 23%, of the variance in protein synthesis. A greater proportion of the variance in protein synthesis was explained by a set of six intracellular amino acids (Leu, Met, Tyr, Gly, Ala, Tau), with R2 = 36%, p = 0.001. Further, multiple regression identified specific combinations of six plasma amino acids which best explained (“predicted”) the levels of each intracellular amino acid predictor of protein synthesis (R2 = 0.4-0.5, p<0.001- 0.0001). Activities of some rate-limiting glycolytic enzymes, pyruvate kinase and phosphofructokinase, were correlated with protein synthesis in the leukocytes (p = 0.036, and 0.002, respectively). Phosphofructokinase, the major regulating enzyme in glycolysis, also was negatively correlated with birth weight and gestational age. These data indicate that protein synthesis is higher in smaller neonates, and that its level can be predicted from levels of a few intracellular amino acids whose concentrations, in turn, can be predicted from specific sets of different plasma amino acids.

699 INTRACELLULAR AMINO ACID LEVELS AS PRE- DICTORS OF PROTEIN SYNTHESIS IN MAN

The relation of protein synthesis (PS), a functional expression of amino acid (AA) balance, to the concurrent intracellular (IC) and plasma (P) concentrations of AA is uncertain. We studied 29 normal young adults & 63 neonates 27-44 weeks post-conceptual age, using the circulating neutrophil (LEUK) as the cell model. PS (3H-LEU incorp, pmoles/hr/mg DNA) in the LEUK, 19 AAs (HPLC, fluorometric detection, nmoles/ml) in both LEUK & P, and cell water content (dry weight-“trapped” water by 14C-inulin) were quantified. In infants, PS was inversely related to birthweight (BWT) & gestational age (GA) (PS = 9265 - 1.2 BWTg -66.35 GA wks;. p=0.0001). With the exception of VAL, CIT, GLN all the ICAA were higher than in P. HIS, ASP, TYR & ORN were the only significant (< .05) self-correlations between 1C & PAA in infants; THR & ILE, in adults. Stepwise multiple regression (MR) was used to identify the set of AAs accounting for the greatest proportion of the variance (R2) in PS. The sets of AA selected by the program were: for PAA in infants, ILE & ALA (R2=0.11, p=0.04); for adults, ASP, GLY, MET (R2)=0.32, p=0.009). For ICAA in infants, LEU, MET, HIS, LYS, GLY, ALA (R2=0.32, p= 0.001); for adults, LEU, MET, HIS, ILE, PHE (R2=0.54, p=0.002). By futher MR analysis, levels of each ICAA predictor of PS in infants & adults was found to be predicted by a different set of PAAs, (e.g., for adults, IC-LEU predicted by P-THR, CIT, VAL, HIS, R2=0.60, p=0.0001). We conclude that: (1) the level of PS can be predicted by a specific set of ICAAs better than by any set of PAAs. (2) The levels of the ICAA predictors of PS can then be predicted by specific, but different, sets of PAAs. These data suggest a possible practical means to modulate cell PS indirectly via ICAA concentrations by altering selected PAA.

1618 CELLULAR MALNUTRITION IN UREMIA

Some of the cellular abnormalities of severe protein-calorie malnutrition in children (PCM), e.g., reduced energy-related enzyme activities, energy and protein synthesis levels and intracellular amino acid deficits, are also thought to occur in chronic uremics. Improvement of these abnormal cell bioactivities, characterizing cellular malnutrition, should stabilize and possibly retard progressive cellular deterioration in uremics. We have used the circulating granulocyte as a cell model to study energy-related enzyme activities (pyruvate kinase (PK), phospho-fructokinase (PFK), adenylate kinase (AK), energy charge (Ech=ATP+0.5ADP/(ATP + ADP + AMP)), protein synthesis (3 H-leucine incorp.) and amino acid pools in 52 adult uremics (9 not dialyzed (ND), 40 hemodialyzed (HD) and 13 peritoneally dialyzed (CAPD)) and 61 normal control (C) subjects. Gross clinical estimators of chronic malnutrition (Wt/Ht ratio, skinfolds, arm muscle area, plasma albumin and total lymphocyte count) were within normal limits for the dialyzed uremics. In the ND uremics, all clinical and cell measures were abnormal. In the HD uremics, cell PK, AK, ATP, Ech and PS were significantly (p < .05) better than ND but less than C. A significant reduction in branched-chain amino acids (BCAA=valine, leucine, isoleucine) and methionine (MET) were noted in both ND and HD patients. In the CAPD patients, after 18 ±9 (SD) months of dialysis PK, PS, BCAAs and MET levels were normalized. Ech was 90% of normal and >HD, but ATP and AK were not improved. We conclude that cellular malnutrition occurs in apparently stabilized adult uremics without clinical signs of malnutrition. CAPD seems to improve many cell bioactivities, and therefore, cellular malnutrition in uremics.

PROTEIN SYNTHESIS, ENERGY LEVEL AND CELL AMINO 1617 ACID IMBALANCE IN CHRONIC UREMICS

To improve protein synthesis (PS) and Energy charge (Ech) should require correction of imbalanced intracellular amino acid levels. In 40 adult chronic uremics stabilized by hemodialysis, multiple regression analysis identified combinations of cell amino acid levels predicting intracellular PS (3 H-Leucine incorp) (PS=10.8 + 0.02 ASP -0.127 VAL + 0.54 ILE + 0.032 ORN -0.12 LYS -0.10 TRP (R2= 0.40, p=0.019)) and Energy charge (Ech=ATP + 1/2 ADP/ATP + ADP + AMP) (Ech (x 103) = 766 -0.75 ASP + 0.18 GLU + 0.59 GLY + 0.53 ORN -0.33 ARG (R2=0.33, p=0.036)). PS and Ech were reduced compared to 32 normals. The leukocyte (∼90% Pm neutrophils) was used as the cell model. 18 adult chronic uremics, stabilized on 3x/wk hemodialysis, were infused with 500ml 10% amino acid solution after each dialysis, dialyzer detached, for 3 months, receiving in toto ≅ 1.5-1.9kg synthetic amino acids. Baseline studies showed decreased (p<.05) PS, Ech and in vitro glycolytic flux (glucose → lactate). The amino acid infusions led to decreased blood pressure and plasma cholesterol level, but did not significantly improve cell bioactivities. The intracellular amino acid imbalance worsened: valine, isoleucine, leucine, methionine, lysine and ornithine were reduced; arginine + glycine were increased. Only tyrosine and phenylalanine levels normalized. We ascribe failure to correct cell bioactivities to failure to correct the cell amino acid imbalance. The leukocyte is a useful cell model to formulate and evaluate therapies to correct abnormal cell metabolism in uremics.

A CELL MODEL TO ASSESS PROTEIN SYNTHESIS AND AMINO ACID POOLS IN NEONATES

The relation of protein synthesis (PS) to extracellular (EC) and intracellular (IC) amino acid (AA) pools, birthweight, gestational age and postnatal weight change in human neonates is unknown. Viable neutrophils isolated from 1-2 ml venous blood were used as a cell model to evaluate these relationships in 35 infants with complete data at 33 to 44 weeks postconceptual age (2d-14 weeks postnatal age), PS (3H-leucine incorp, p moles/hr/mg DNA) and 19 cell AA (n moles/mg DNA) were quantified in the leukocytes and 19 AAs in the same plasma (n moles/ml) (Dionex high pressure AA analyzer, fluorometric detection). No significant (p <.05) correlations were detected between PS and individual plasma AAs. PS was correlated with 11 of 19 IC AAs and with sets of both essential (ESSAA) and nonessential (NESSAA) IC AAs. PS also was inversely (−) correlated with birthwt., gest.age, postconceptual age and weight at time of study. When studied in a stepwise multiple regression of PS on the age-weight variables, weight at study was the only variable selected by the model and accounted for 20% (R2=.205, p=0.006) of the variance in PS. Of the AAs, the IC ESSA As were the only set explaining a significant proportion of the variance in PS (R2=0.274, p=0.001). Thus it appears that the smaller and more preterm the infant, the greater the rate of protein synthesis; which, in turn, is more closely related to levels of IC ESSA As than to plasma concentrations of the AAs. Measures of protein synthesis and IC AAs are feasible, even in neonates, and should contribute significantly to formulation and evaluation of therapies designed to improve these modalities.

CHOLESTEROL, CAROTENE AND BIRTHWEIGHT IN SMOKING MOTHERS

It has been suspected that nutrition during pregnancy might counteract the adverse effects of smoking on birthweight (BWT). In our previous prospective studies of ∼2100 mothers, 47% were smokers who had 63% of the babies with BWT below our median (3300g). A prospective, randomized, controlled trial of a food supplement (WIC) from 19 weeks to term in 410 of these free-living mothers led to increased mean BWT +91g (p=0.039), which was greater in smokers (+115g, p=0.034), taking into account sex, gestational age, race, prenatal visits. In 288 mother/baby pairs multiple regression analysis revealed an interaction between maternal midpregnancy (19 weeks) plasma levels of cholesterol (chol), β carotene (βC) and smoking on BWT (p=0.017). Prospective data on the interactive effects of βC, chol and smoking on BWT apparently have not previously been noted. High βC (> 116 μg/dl) and low chol levels (< 150 mg/dl) were associated with significantly larger babies (+ ∼500g) in the smokers. If βC levels remained high from 19 to 36 weeks of gestation, heavy smokers delivered large babies. If βC levels decreased, the babies were small. In nonsmokers, no interaction between these nutrients on BWT was detected, but BWT was ∼300g more with higher chol, i.e., from 116 to 272 mg/dl at midpregnancy, at all levels of βC. We conclude that certain plasma levels of β carotene and cholesterol in smokers might counteract the adverse effect of smoking on fetal growth.

271 IMPAIRED FETAL GROWTH FROM AMINO ACID IMBALANCE

Whether fetal growth retardation (FGR) could be induced by diets imbalanced relative to a single amino acid, was investigated. Pregnant rats were fed either a 6% casein “basal” (B) diet supplemented by L-MET & a 5% mixture of essential, plus 4.5% small neutral dispensible amino acids, (4 kcals & 0.16g protein-equivalent/g diet, complete in essential nutrients), an “experimental” (E) diet (same, except 0.4% L-THR supplement omitted) or a “control” (C) diet, like B, but containing 20% casein. 343 fetuses with placentas were delivered by Caesarian section usually on day 21 but occasionally on day 20 or 22. Food consumption was similar in all groups (kcals/100g rat/d): protein intake of the B & E rats was about half that of the C group. Net maternal weight gain (excluding conceptus) was +8% in C, 0% in B & −12% in E group dams. Body weight, length, volume, brain & placental weights, adjusted by multivariate analysis to control for maternal prepregnant weight, length, litter size, net weight gain & days of gestation, were significantly reduced in 156 B vs 32 C & in 155 E vs B fetuses. Net maternal weight loss did not prevent FGR. Of the total variance in birth measures, protein restriction accounted for 30-59% in the B vs C group fetuses; & the THR amino acid imbalance for 36% between the E & B groups. Thus, diets with excess dispensible amino acids during pregnancy induce FGR, especially when THR is limiting.

481 MATERNAL SMOKING MAY IMPAIR EETAL NUTRITION

Cigarette smoking by pregnant women is known to significantly reduce birth weight of their babies. The effect appears dose-related. The mechanism is uncertain. We have shown previously that maternal plasma nutrient & amino acid levels, & some polymorphonuclear leukocyte (PMNL) enzyme & metabolite levels at midpregnancy are closely related to, predictive of, & account for, about 20% of the variance in birth weight of the baby. In a prospective study of ∼600 mother/baby pairs who had complete demographic & biochemical data, 294 mothers were non-smokers, 285 smoked. Birth weight, adjusted for gestational age & sex of the baby, & for race, age, prepregnant height, weight, & parity of the mother, was significantly reduced in proportion to the number of cigarettes reportedly smoked daily. A two-tailed t test showed that smoking mothers had significantly decreased PMNL G6PDH activity, ATP & ADP levels. Hair root protein content was reduced, as were plasma total protein, albumin, β & γ globulins, & cholesterol. The levels of plasma free amino acids ALA, CYS & LYS also were significantly reduced. Analysis of variance showed significant differences between standardized scores for nonsmokers & those who smoked 1-10, 11-20, 21-30, > 30 cigarettes/d for education, hematocrits, diet calorie & protein intakes (recall), PMNL G6PDH & ATP, hair root protein & DNA, plasma total protein, α 2, β & γ globulin, & the amino acids: GLU, LYS, HIS, & ORN. We conclude that smoking during pregnancy alters maternal plasma nutrient levels, protein stores & cell PMNL metabolism. These nutritional effects might account for the reduced birth weight.

1525 CELL FUNCTIONS IN UREMIA IMPROVED BY DIALYSIS AND AMINO ACID INFUSIONS

Chronic uremia is often associated with impaired utilization of glucose, presumably intracellularly, since it is not dependent on insulin or insulin redeptors. Using the circulating polymophonuclear leukocyte (PMNL) as a cell model, 9 nondialyzed & 26 stable, maintenance hemodialyzed adults with chronic uremia (residual Ccr < 5ml/min) had significantly reduced levels of glycolytic enzyme activities pyruvate kinase (PK) & glucose -6-PO4 dehydrogenese (G6PDH), ATP, & adenylate kinase (AK), protein (3 H Leu) & RNA (3H-URI) synthesis, compared to 32 normal control subjects (blood donors). Following in vitro incubation of isolated PMNL in Krebs-Henseleit medium with 5mM glucose, cell levels of the glycolytic metabolites G6P, F6P, & F-1-6-P were reduced, while DHAP, 3-PGA & PEP were increased, indicating a perturbation in glycolytic flux (GF), although lactate production was not impaired. Hemodialysis improved AK activity & protein synthesis (PS), but G6PDH & ATP decreased. Lactate production by incubated PMNL fell. The postdialysis changes reverted to predialysis values within 1 week. Stable maintenance hemodialysis patients got 500ml amino acid infusions (AAI) (Aminosyn 10%) at the end of each 3×/week dialysis. After 12 infusions (1 month), predialysis ATP levels & PS reached normal levels. GF lactate production was unaffected but triose phosphate levels increased. To the extent that PMNL s reflect cell metabolism, the data is consistent with a cellular defect in glycolysis & protein synthesis in chronic uremics. Dialysis, coupled with AAI, improves cell functions.