Analysis of 3-hydroxydodecanedioic acid for studies of fatty acid metabolic disorders: preparation of stable isotope standards

Current diagnostic tests to detect disorders of fatty acids metabolism, such as long-chain hydroxyacyl CoA dehydrogenase deficiency (LCHAD), are hampered by insensitivity or a long delay time required for results. Children with LCHAD deficiency are known to excrete 3-hydroxydicarboxylic acids with chain lengths of 10-16 carbons, but a quantitative method to measure excretion of these potentially diagnostically important compounds has not been reported. We report synthetic schemes for synthesis of 3-hydroxydodecanedioic acid and a di-deuterated analog, suitable for use in a stable-isotope dilution mass spectrometric analytical approach. Evaluation of several common derivatization protocols to produce a volatile derivative for gas chromatography determined that trimethylsyl derivatives produced the best efficiency and stability. Positive-ion chemical ionization mass spectrometry provided the greatest yield of characteristic ions. These results indicate the basic reagents needed to develop sensitive and accurate 3-hydroxydodecanedioic acid measurements for diagnosis of LCHAD deficiency and other fatty acid oxidation disorders.     

Twin studies in metabolic diseases.

The traditional role of twin studies has been to assess the relative role of genetic factors as a first step in defining the genetic architecture of complex traits. This has been based on the realization that monozygotic pairs (MZ) share all their genes, while dizygotic pairs (DZ) share 50% of their genes on average. Thus, greater similarity of MZ pairs compared to DZ pairs has been taken as prima facie evidence of the role of genetic factors. This is true provided the environmental similarity of MZ pairs is not greater than for DZ pairs for effects relevant to the trait in question. This first step in genetic studies was carried out long ago in many research areas, but not in others. More detailed knowledge of the genetic architecture of traits is then obtained by other means. In this paper, we give a brief overview of some results for metabolic diseases (ischaemic heart disease, hypertension, subarachnoid haemorrhage, NIDDM and IDDM) using the classical twin approach in a large, unselected population-based twin cohort. We also outline approaches to using twins that we believe will continue to be useful, particularly for the study of environmental effects.     

Association of kidney function and metabolic risk factors with density of glomeruli on renal biopsy samples from living donors

OBJECTIVE: To test the hypothesis that kidney function and metabolic risk factors are associated with glomerular density on renal biopsy samples from healthy adults.PATIENTS AND METHODS: This study compared glomerular density with predonation kidney function, blood pressure, and metabolic risk factors in living kidney donors at Mayo Clinic in Rochester, MN, from May 10, 1999, to February 4, 2009. During implantation of the kidney allograft, an 18-gauge core needle biopsy sample of the renal cortex was obtained, sectioned, and examined by pathologists. Glomerular density was determined by the number of glomeruli (normal and sclerotic) divided by area of cortex.RESULTS: The study sample of 1046 kidney donors had a mean of 21 glomeruli (0.8 sclerotic glomeruli) and a glomerular density of 2.3 glomeruli per square millimeter. In a subset of 54 donors, glomerular density inversely correlated with the mean glomerular area (r_s=−0.28). Independent predictors of decreased glomerular density were older age, increased glomerular filtration rate, family history of end-stage renal disease, increased serum uric acid, and increased body mass index. Increased urine albumin excretion, hypertension, decreased high-density lipoprotein cholesterol, and metabolic syndrome were also associated with decreased glomerular density after age-sex adjustment. These associations were not explained by the presence of glomerulosclerosis, tubular atrophy, interstitial fibrosis, or arteriosclerosis on the renal biopsy sample. In older donors, decreased glomerular density was attenuated by an increased prevalence of glomerulosclerosis and tubular atrophy.CONCLUSION: Decreased glomerular density is associated with many different kidney function and metabolic risk factors among relatively healthy adults and may represent an early state of increased risk of parenchymal injury.BMI = body mass index; CKD = chronic kidney disease; ESRD = end-stage renal disease; GFR = glomerular filtration rate; HDL = high-density lipoproteinThe size of individual nephrons can reflect important elements of metabolic regulation. Persons with a low nephron endowment or with certain stress states (eg, obesity, pregnancy, or unilateral nephrectomy) develop glomerular hypertension and increased single-nephron filtration (hyperfilter) with compensatory glomerulomegaly.^1-5 Glomerulomegaly from hyperfiltration also occurs in response to nephron loss, perhaps due to a shift in perfusion from nonviable to viable nephrons.^6,7 In addition to glomerulomegaly, hyperfiltration leads to tubular hypertrophy and hyperplasia.^8-10 Much of the volume increase of viable nephrons in response to hyperfiltration may be in the proximal tubule rather than the glomerulus.^11-13 Unfortunately, there is no safe and practical method to directly measure the average volume occupied by nephrons in living humans.Normal kidney parenchyma consists only of nephrons and supporting vessels, with a trivial amount of interstitium (Figure 1), and thus glomerular density on sectioned biopsy samples of renal cortex is inversely proportional to average nephron size to some extent. We hypothesized that glomerular density is associated with kidney function and metabolic characteristics of the kidney. Specifically, among persons with risk factors for glomerular hypertension and hyperfiltration, the glomerular density would be decreased. The rationale for this hypothesis is that any process that increases the volume occupied by each nephron would effectively push glomeruli apart, leading to decreased glomerular density. For example, decreased glomerular density has been associated with low birth weight in neonates and, in this instance, has been attributed to low nephron endowment with compensatory hypertrophy.¹⁴ Alternatively, kidney function and metabolic risk factors may be associated with global glomerulosclerosis (complete scarring of glomerulus), and reabsorption of these sclerotic glomeruli¹⁵ could decrease glomerular density.Open in a separate windowFIGURE 1.Representative fields of implantation biopsies (original magnification x100; hematoxylin-eosin) for kidney donors with (top) high glomerular density (10 glomeruli in a representative field of a biopsy section) and (bottom) low glomerular density (2 glomeruli in a representative field of a biopsy section). G = glomerulus.For editorial comment, see page 271In the current study, we used adult living kidney donors with implantation (intraoperative) renal biopsy samples to study glomerular density. Although kidney donors are selected on the basis of good health, they still demonstrate substantial variation in kidney function, blood pressure, and metabolic profiles.¹⁶ Our goal was to compare glomerular density against predonation clinical characteristics, particularly those that have previously been implicated in glomerular hyperfiltration and glomerular hypertension or are risk factors for chronic kidney disease (CKD).     

An in vitro human muscle preparation suitable for metabolic studies. Decreased insulin stimulation of glucose transport in muscle from morbidly obese and diabetic subjects.

We have developed an in vitro muscle preparation suitable for metabolic studies with human muscle tissue and have investigated the effects of obesity and non-insulin-dependent diabetes mellitus (NIDDM) on glucose transport. Transport of 3-O-methylglucose and 2-deoxyglucose was stimulated approximately twofold by insulin in muscle from normal nonobese subjects and stimulation occurred in the normal physiological range of insulin concentrations. In contrast to insulin stimulation of 3-O-methylglucose and 2-deoxyglucose transport in muscle from normal, nonobese subjects, tissue from morbidly obese subjects, with or without NIDDM, were not responsive to insulin. Maximal 3-O-methylglucose transport was lower in muscle of obese than nonobese subjects. Morbidly obese patients, with or without NIDDM, have a severe state of insulin resistance in glucose transport. The novel in vitro human skeletal muscle preparation herein described should be useful in investigating the mechanism of this insulin resistance.     

Plasma ascorbic acid preparation and storage for epidemiological studies using TCA precipitation

Objectives:To introduce a procedure to validate an ascorbic acid method using trichloroacetic acid (TCA) for plasma stabilization at different storage temperatures.Methods:EDTA and heparin plasma were precipitated with TCA (1:5) containing 0.54 mol/L EDTA, or without. Samples were stored at ? 20 °C and ? 70 °C and their stability was tested at room temperature for 24 h.Results:A significant 40% loss (p < 0.001) of plasma ascorbic acid was found when EDTA samples with added EDTA were stored at ? 20 °C for 2–4 weeks compared with storage at ? 70 °C. Ascorbic acid in heparin plasma without added EDTA was most unstable and samples left at room temperature for 24 h lead to almost a total loss of ascorbic acid. Addition of EDTA to the TCA solution improved stability of samples of both plasma types at room temperature.Conclusion:The recommended procedure for ascorbic acid determination in plasma stabilized with TCA is immediate storage at ? 70 °C and inclusion of EDTA into the TCA solution.     

Mechanistic studies on metabolic interactions between gemfibrozil and statins

A series of studies were conducted to explore the mechanism of the pharmacokinetic interaction between simvastatin (SV) and gemfibrozil (GFZ) reported recently in human subjects. After administration of a single dose of SV (4 mg/kg p.o.) to dogs pretreated with GFZ (75 mg/kg p.o., twice daily for 5 days), there was an increase (approximately 4-fold) in systemic exposure to simvastatin hydroxy acid (SVA), but not to SV, similar to the observation in humans. GFZ pretreatment did not increase the ex vivo hydrolysis of SV to SVA in dog plasma. In dog and human liver microsomes, GFZ exerted a minimal inhibitory effect on CYP3A-mediated SVA oxidation, but did inhibit SVA glucuronidation. After i.v. administration of [(14)C]SVA to dogs, GFZ treatment significantly reduced (2-3-fold) the plasma clearance of SVA and the biliary excretion of SVA glucuronide (together with its cyclization product SV), but not the excretion of a major oxidative metabolite of SVA, consistent with the in vitro findings in dogs. Among six human UGT isozymes tested, UGT1A1 and 1A3 were capable of catalyzing the glucuronidation of both GFZ and SVA. Further studies conducted in human liver microsomes with atorvastatin (AVA) showed that, as with SVA, GFZ was a less potent inhibitor of the CYP3A4-mediated oxidation of this drug than its glucuronidation. However, with cerivastatin (CVA), the glucuronidation as well as the CYP2C8- and CYP3A4-mediated oxidation pathways were much more susceptible to inhibition by GFZ than was observed with SVA or AVA. Collectively, the results of these studies provide metabolic insight into the nature of drug-drug interaction between GFZ and statins, and a possible explanation for the enhanced susceptibility of CVA to interactions with GFZ.     

靶向超声造影剂制备的方法学研究

靶向超声造影是目前超声造影的前沿性课题，随着超声造影剂在临床的不断应用与实践，使得超声诊断学与治疗学发生了跳跃式的前进。靶向超声造影技术通过靶向作用于生物分子组成成分来突出显示病变组织的病变部位，从而提高影像诊断的准确性与敏感性，因此靶向超声造影剂成为现今研究领域的热点。然而，要想设计生产靶向超声造影剂能够按照预定的靶向至病变部位，且能流经循环系统后依然保持其特有的稳定性，故要求研究靶向超声造影剂的学者不断在基础研究与方法学上改进。现就靶向超声造影剂的方法学方面作一综述。