Derivatives of human growth hormone (hGH) of increasing size were produced by reaction with the Nhydroxysuccinimide ester of polyethylene glycol-5000 (PEG 5000 ), a 5-kDa reagent that selectively conjugates to primary amines. By adjusting the reaction conditions and purification procedure, it was possible to isolate hGH derivatives containing up to seven PEG moieties that altered the Stokes radius and thereby the effective molecular masses of the unmodified hormone from 22 to 300 kDa. Fortunately, the most reactive amines were ones that did not lie in either of the two sites important for receptor binding. Nonetheless, increasing the level of PEG modification linearly reduced the affinity of hGH for its receptor and increased the EC 50 in a cellbased assay up to 1500-fold. Most of the reduction in affinity was the result of slowing the association rate for the receptor. The clearance rate of hGH in rats was inversely proportional to effective molecular weight and closely fit a filtration model. We have tested the potency of these analogs by injecting them daily or every 6 days into hypophysectomized rats and determining the effects on body and organ growth. The efficacy of these analogs was optimal for hGH conjugated with 5 eq of PEG 5000 , and the potency was increased by about 10-fold compared with unmodified hGH. Such PEG-hGH derivatives show promise as long-acting alternatives to daily injections of hGH. More generally these studies show that improving hormone clearance properties, even at the expense of reducing receptor binding affinity, can lead to dramatic increases in hormone efficacy.The ability of a hormone to elicit a biological effect in vivo depends on many factors including the affinity for its receptor and the rate at which it is cleared from the circulation. Some hormones, like atrial natriuretic peptide, have a very high affinity for their receptor (10 pM) and are cleared very rapidly (t1 ⁄2 ϳ0.5 min) by receptor and protease-mediated events (1). Other hormones, like human growth hormone (hGH), 1 have lower affinity for their receptor (300 pM) but are cleared more slowly (t1 ⁄2 ϳ30 min in rats), primarily via the kidney (2, 3).Understanding the relationships between hormone affinity, clearance, and efficacy is important in optimizing hormone therapy. To study this systematically one would like to vary these parameters and evaluate their relative importance in regulating biopotency. hGH is a good model system in this regard as much is known about its structure and function (for review see Ref. 4). Simple receptor binding (5, 6), cell-based assays (7,8), and growth parameters in rodents (9) can be used to determine biopotency in vitro and in vivo. The properties of proteins such as hGH that are cleared by kidney filtration can be modulated by attachment of polyethylene glycol (PEG) polymers, which increases the hydrodynamic volume of the hormone and thereby slows its clearance (10, for recent review see Ref. 11).Here, we describe a set of hGH derivatives conjugated with increasing numbers of PEG ...

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RENATAL DIAGNOSIS IS USEFULfor managing a pregnancy with an identified fetal abnormality and may allow for planning and coordinating care during delivery and the neonatal period. 1 A variety of prenatal diagnostic tests are available but have limitations. Noninvasive tests such as maternal serum marker testing and ultrasound can be used to screen for the presence of chromosomal abnormalities but are not definitive. [2][3][4][5] On the other hand, invasive diagnostic tests (eg, amniocentesis, chorionic villus sampling, percutaneous umbilical blood sampling) for fetal chromosomal abnormalities are highly reliable, but the procedure used for each test carries a risk for loss of pregnancy. 6,7 Many patients who are candidates for these tests decline them because of the risk of pregnancy loss.An alternative to existing methods for prenatal diagnosis is to use fetal cells and fetal DNA that exist in the maternal circulation. [8][9][10][11][12][13][14][15] Circulating fetal DNA has been used to determine the sex of the fetus through detection of sequences present on the Y chromosome. 13 In addition, several studies have

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The hierarchy of diet components (e.g., protein, carbohydrate, vitamins, and minerals) influencing growth hormone (GH), insulin-like growth factor-I (IGF-I), and their binding proteins (BP) is not well defined. Young adult rats were fed diets for 1 mo that included low protein or 60% and 40% of carbohydrate calories. We hypothesized that levels of both hormones, their dominant BPs and liver IGF-I mRNA would fall, and that part of the mechanism for decreasing serum IGF-I would be enhanced IGFBP-3 protease activity. By day 30, caloric deprivation to 40% lowered serum GH, GHBP, IGF-I and IGFBP-3, and liver IGF-I mRNA. This was the only condition resulting in body weight loss (-15%) vs 39% gain in controls. Restriction to 60% calories had no impact on BP levels, slightly lowered IGF-I ( -12% ) in the face of a 95% inhibition of GH levels, while allowing a modest 9% body weight gain. Protein deprivation lowered serum GH, IGF-I and IGFBP-3, and liver IGF-I mRNA, while GHBP levels were normal. The reduced total IGF-I under these dietary conditions could not be explained by an increase in IGFBP-3 protease activity, or a decrease in the association of IGF-I with IGFBP-3 and the acid labile subunit. (J. Clin. Invest. 1995Invest. . 95:2258Invest. -2265