William S. Stirewalt

Angiotensinogen genotype and plasma volume in nulligravid women

Objective To determine if nonpregnant plasma volume is altered in women who are homozygous for the T 235 coding angiotensinogen allele, which predisposes women to an increased risk of preeclampsia. Methods We measured plasma volume by Evans blue dilution and analyzed it as a function of angiotensinogen genotype in 15 nulligravid women during midfollicular phase of 26 menstrual cycles. Eleven women were evaluated during two cycles, and four women were evaluated in one cycle. Fourteen women were white, and one was Asian. No subjects had illnesses or were taking medication. The range of body mass index (BMI [kg/m2]) was 20.2-31.0. Plasma volume (mL) was reported as plasma volume divided by BMI to control for variations in body sizes. Statistical analysis was performed by analysis of variance with post hoc testing using Fisher least significant difference test for multiple comparisons (P < .05 accepted for significance) Results Angiotensinogen genotype analysis showed five women homozygous for M 235, three women homozygous for T 235, and seven women who were heterozygous (MT 235). T 235 homozygotes had significantly lower plasma volume divided by BMI compared with women who were homozygous for M 235 and women who were heterozygous for MT 235 (mean 1 standard deviation [SD] [71.2 + 8.8, 86.6 + 5.2, 95.8 + 15.6, respectively, P < .05]). There was a tendency toward higher plasma volume in heterozygote MT 235 compared with homozygote M 235 carriers, but it was not statistically significant. Conclusion We conclude that the homozygous T 235 coding angiotensinogen genotype is associated with reduced plasma volume in nulligravid women during the follicular phase of the menstrual cycle compared with M 235 homozygotes and heterozygotes. This association of the T 235 coding genotype might contribute to fetal growth restriction in preeclampsia.

Amniotic fluid glycine-valine ratio and neonatal morbidity in fetal growth restriction.

Objective To test the hypothesis that an elevated amniotic fluid glycine-valine ratio predicts neonatal morbidity in growth-restricted newborns. Methods Amniotic fluid (AF) was collected from 122 third-trimester pregnancies (range 31–39 weeks), 49 of which were complicated by fetal growth restriction. Amino acid analysis was performed by high-pressure liquid chromatography. Glycine-valine ratios were compared between normal and growth-restricted fetuses. Neonatal morbidity within the group of growth-restricted fetuses was characterized by evaluation of neonatal hypoglycemia, arterial cord blood gas analysis, and birth weight percentile. We also examined the correlation of AF glycine-valine ratio to the umbilical artery resistance index. The median interval between AF sampling and delivery was 1 day (range 0–8 days). Analyses were performed by Student t test X2 with Yates correction, or simple correlation when appropriate. P < .05 was considered significant. Results Growth-restricted fetuses have a significantly elevated AF glycine-valine ratio compared with control subjects (3.31 ± 1.06 versus 2.61 ± 0.77, respectively, P < .001). There was no association of the glycine-valine ratio with gestational age for either group. An elevated glycinevaline ratio was not associated with neonatal hypoglycemia within the growth-restricted group (hypoglycemia: [n = 16] 3.19 ± 1.07; no hypoglycemia; (n = 30) 3.44 ± 1.09). There were no significant correlations of glycine-valine ratio with arterial cord blood pH (r = −0.10), oxygen pressure (r = 0.04), or base deficit (r = 0.12). There were no significant correlations of glycine-valine ration and birth weight percentile (r = −.24) or umbilical artery resistance index (r = −.14). Conclusion Amniotic fluid glycine-valine ratio is elevated in growth restricted compared with control fetuses. However, he level of glycine-valine elevation is not associated with neonatal morbidity related to hypoglycemia, arterial cord blood gas abormalities, or birth weight percentile.

Comparison of the use of isotopic proline vs leucine to measure protein synthesis in cultured fibroblasts

Compartmentation of the amino acid precursor pools for protein synthesis in cultured cells can substantially complicate measurements of synthesis rates. This is particularly true for nonessential amino acids such as proline, an amino acid often used in isotopic form to measure collagen synthesis. We have made a comparative study of this problem in cultured IMR-90 fibroblasts using isotopic proline and leucine to measure total protein and collagen synthesis. 3H-leucine in the extracellular (EC) medium equilibrates with tRNA-leucine at an EC concentration of 0.4 mM in both dividing and stationary cells. Thus, under these experimental conditions there is no complicating compartmentation of leucine for protein synthesis. Equilibration of EC and tRNA-bound 3H-proline, however, does not occur even when the EC concentration is in the mM range, based upon simultaneous measurements of synthesis rates using 3H-proline and 3H-leucine together. Furthermore, significant changes in EC proline concentration and specific activity occur over short time intervals (2 hr) if the initial EC proline concentration is below 0.2 mM. Thus, the use of isotopic proline to measure protein synthesis introduces substantial interpretive problems. Serum deprivation causes changes in both total collagen synthesis and the percent of protein synthesis devoted to collagen when measured with either 14C-leucine or 3H-proline. At the same time, isotopic proline remains the better choice for measuring percent collagen synthesis.

Hemodynamic and Biochemical Characteristics of the Aorta in the WKY, SHR, WKHT, and WKHA Rat Strains

This study was designed to characterize the hemodynamic and biochemical properties of the abdominal aorta in four genetically related inbred rat strains that express genetic hypertension and hyperactive behavior in varying combinations. These include (1) the spontaneously hypertensive rat (SHR), which is hypertensive, hyperactive, and hyperreactive to stress; (2) Wistar-Kyoto (WKY) rats, which express none of these traits; (3) WKHT rats, which are hypertensive but not hyperactive; and (4) WKHA rats, which are hyperactive and hyperreactive to stress, but normotensive. Together, these four strains allowed us to examine the structural and functional changes in the aorta in the hypertensive SHR, the most widely used animal model of genetic hypertension, while controlling for the variables of hyperactivity and hyperreactivity that are also expressed in the SHR. Four groups of animals of both sexes were studied: (1) WKY, n = 101, (2) WKHA, n = 33, (3) WKHT, n = 91, and (4) SHR, n = 28. Blood pressure (BP) was determined by tail plethysmography as well as direct intraarterial monitoring under anesthesia. Fixed specimens were prepared for histologic analysis and the wall thickness determined morphometrically. Quantification of soluble tissue protein, elastin, and collagen in the aortic tissue was determined by measuring leucine (leu), hydroxyproline (HP/leu), and desmosine (DES/leu). The hypertensive strains (SHR and WKHT) had significantly higher tail BP than the normotensive strains (WKY and WKHA)-WKY: 128.7 +/- 22.3; WKHA: 126.7 +/- 14.6; WKHT: 162.8 +/- 21.2; SHR: 164.2 +/- 36.1 (p < 0.0001). Additionally, intraaortic diastolic BP and mean BP were higher in SHR rats than in WKHT. Morphometric studies showed the media thickness in the SHR rats was significantly greater than in the WKY and WKHA rats and no different than in the WKHT rats. Significantly less of the aortic wall protein was present as elastin in the hypertensive rats (SHR and WKHT), as well as the hyperactive rats (WKHA), compared to rats that had neither trait (WKY). These studies provide new information regarding aortic structure and function in genetic hypertension using inbred strains to control for the hyperactivity/hyperreactivity traits that coexist with hypertension in the SHR. They reveal that hypertensive aortas have altered matrix proteins that cannot be explained simply on the basis of blood pressure alone.

Amniotic Fluid Glycine/Valine Ratio Is Elevated in Fetuses with Growth Retardation

Alterations in the amnio acid content of fetal and neonatal cord blood characteristic of chronic starvation have been identified in growth-retarded offspring. We hypothesized that these changes in amino acid patterns, specifically an increase in the glycine/valine ratio, would be present in the amniotic fluid of growth-retarded fetuses. Amniotic fluid specimens from 63 singleton pregnancies in the third trimester (range 33-39 weeks) were examined. Twenty-five pregnancies were complicated by fetal growth retardation (FGR) which was suspected by ultrasound and confirmed at birth. Amniocenteses were performed for routine clinical indications. Amino acid analysis was performed on prepared amniotic fluid specimens by high-pressure liquid chromatography. The glycine/valine ratio in amniotic fluid as a function of gestational age was compared between growth-retarded and control (C) groups by analysis of covariance. Amniotic fluid from growth-retarded fetuses demonstrated a significantly higher glycine/valine rat...