Higher Catabolic Rate Research Articles

Diet and Gender Influence Survival in Sickle Cell Disease

Increased childhood mortality and reduced longevity persist in the resource-limited settings with the highest populations of sickle cell disease (SCD) (Piel et al., New Engl J Med 2017). A high catabolic rate caused by multiple phenomena including compensatory erythropoiesis suggest increased calorie and nutritional requirements for individuals with SCD; however, a vast majority of individuals with SCD live under malnourished conditions. Also, it is unknown if the survival of offspring is influenced by the parents' diet and if the diet of individuals has a gender-specific effect on their survival.To address this critical unmet need for information under controlled conditions, we performed a randomized double-blind trial on transgenic Berkley mice with SCD comparing an enriched 'sickle diet' (SD) with a standard 'rodent diet' (RD). The study was performed following protocols approved by the Institutional Animal Care and Use Committee. Breeder pairs comprised of hemizygous (HbAS) females crossbred with homozygous (HbSS) male mice were divided into two dietary groups: [i] SD (59M3, TestDiet; St. Louis, MO) and [ii] RD (9F, 5020, Harlan Laboratories; Madison, WI). SD contained 26.4% protein, 11.1% fat, 27.5% kcal from protein, and 26% kcal from fat. RD contained 18.6% protein, 6.2% fat, 24% kcal from protein, and 18% kcal from fat. SD had a higher mineral, vitamin, amino acid, and omega-3 fatty acid content than RD. Pups were weaned 3 weeks after birth and were then genotyped and phenotyped to select HbSS pups. Pups were then fed SD for 10 days and randomly assigned to either [i] SD or [ii] RD for the remainder of the study. Their survival was followed until the pups were 150 days of age. Analysis was performed on the relationship between rates of survival and the following factors of interest: pup gender, pup diet, and parental diet. The outcome variable of interest, survival in days, was examined using (1) Kaplan-Meier plots of estimated survival and (2) Cox regression to obtain hazard ratios (HR). For Cox regression, the significance of individual factors adjusted for multivariate data and the significance of overall differences were based on Wald tests and log-rank tests respectively. Data were analyzed using the package 'survival' in ' R 3.4.0 ' (R Foundation for Statistical Computing; Vienna, Austria). P <.05 was considered significant.Of the 601 pups, 482 were on SD and 119 were on RD. After 150 days, the overall survival percentages of pups on SD and RD were 77% and 51% respectively. Pups fed SD had significantly prolonged survival rates compared to pups on RD when adjusted for multivariate data (p = 6.03*10-8, HR: .3988, 95% confidence interval (CI): .2860 - .5561). Cumulatively, there were 338 female pups and 263 male pups. After 150 days, the overall survival percentages of female and male pups were 76% and 68% respectively. Gender significantly independently impacted survival with females showing greater survival than males when adjusted for multivariate data (p = .0125, HR: 1.4797, 95% CI: 1.0879 - 2.0126). After 150 days, the overall survival percentages of the 488 pups with parents on SD and the 113 pups with parents on RD were 73% and 68% respectively. Pups born to parents fed SD showed a trend towards increased survival; however, parental diet did not have a statistically significant impact on the survival of pups adjusted for multivariate data (p = .5392, HR: .8878, 95% CI: .6071 - 1.2982). Notably, male pups on RD born to parents on RD showed the poorest survival compared to all other groups, and male pups on SD born to parents on RD showed a significantly improved survival in comparison (p=0.0228), suggesting that improving the individual's diet can overcome the effect of the parents' diet.A diet rich in protein, fat, and micronutrients significantly prolonged the survival of HbSS mice overall. HbSS females survived significantly longer than HbSS males. HbSS mice born to parents fed SD showed a trend towards increased survival; however, parental diet did not have a statistically significant impact on the survival of female pups. Overall, the data emphasized the importance of parents' diet, individual's diet, and gender in SCD. We speculate that an improvement of diet may reduce manifestations of SCD including pain, infections, inflammation, and organ damage and result in increased survival. These data also suggest the consideration of gender and diet in studies using mice with SCD to improve translational outcomes. DisclosuresGupta:Fera Pharmaceuticals LLC: Consultancy, Honoraria; Tautona Group: Consultancy, Honoraria.

Method for the Detection of Insulin‐independent Glucose Uptake in Skeletal Muscle

The quantification of glucose uptake into tissues, alongside co‐solutes flux, is essential to advance basic knowledge of glucose transport and metabolism. However, comprehensive understanding of glucose metabolism has been elusive. A major player in this crucial process is muscle tissue ‐ its large mass, high catabolic rate and storage capabilities make a major contribution to glucose homeostasis. The traditional insulin‐dependent model has proven insufficient to explain a well‐documented clinical observation: Type II diabetic, insulin resistant patients can lower their blood sugar levels with frequent exercise. One challenge in studying a complex mechanism like glucose uptake in muscles is the lack of experimental tools with multi‐channel capabilities, able to quantify most of, if not all the possible related solute fluxes within current models, in‐vivo or ex‐vivo. Our goal was to develop a method to measure glucose, K+, Na+, Ca2+, Fe, Cu, Zn2+ and Mn flux in biological samples using Inductively Coupled Plasma – Mass Spectroscopy (ICP‐MS). ICP‐MS is well established as a sensitive and versatile tool for direct detection and quantification of specific elements.In this study, we developed a new ratiometric method, based on stable‐isotope dilution, to quantify 13C‐based molecules using the molar ratio of 13C/12C. This, added to the multi‐metal detection capabilities of ICP‐MS was combined for a multi‐channel ion flux measurement in mouse EDL muscle during contraction‐stimulated glucose uptake. The preliminary results demonstrated a correlation of K+ uptake with glucose uptake. We showed involvement of the Na+K+ATPase (NKA) with this process using ouabain. An isolated EDL muscle was either stimulated at 90 Hz for 5 min or left unstimulated in 11mM 13C glucose uptake solution at 32°C. The muscle was then washed at 0°C for 20 minutes to wash away excess glucose on the surface of the muscle and to inhibit transporter activity. The muscle was then digested in 1:1 HCl and H2SO4 to break it down to inorganic forms. Finally, we used ICP‐MS to quantify glucose and co‐solute transport. The transport rate for glucose (nM/g min) for stimulated tissues was 601 as compared to 9 for unstimulated tissues. The stimulated muscles also took up K+, as measured through Rb+ tracer, indicating that NKA was active during contraction‐stimulated glucose uptake. To determine which isoform of Na,K,ATPase (NKA) the glucose was coupled to, we used 0.75uM ouabain. This concentration is enough to maintain muscle contractions as well as inhibit NKA α2 but not α1. Ouabain reduced the average glucose uptake rate by 81 %. These preliminary results show that NKA α2 may be coupled to a possible novel glucose transporter in skeletal muscle tissue or to GLUT‐4 in an undescribed way. The development of a method to measure glucose concentrations in biological samples together with coupled fluxes of other ions will advance knowledge of glucose handling in health and disease. In addition, our method to reproducibly quantify C‐based organic molecules by ICP‐MS will advance applications of ICP‐MS to many other fields of biomedical research.Support or Funding InformationNIH RO1 AR 063710

Impact of Spirulina Supplementation on Triglyceride and Serum Cholesterol Level of HIV Infected Patient

Dyslipidemia is common syndrome seen in HIV infected patient due to various reasons like adverse effect of drug, higher catabolic rate, reduced functioning of body organs and reduced immune functioning which contributes adversely to overall cardio-vascular risk profile of the patient. Hence, study was under take to determine the impact of Spirulina supplementation on the lipid profile of HIV infected patients. Patients were followed up at regular interval of two weeks to assure that they take regular Spirulina supplementation, to develop good rapport and to stop dropout rate. During course of study patients were advised to continue their medical prescription. Data was compiled and the results were analyzed using suitable software and appropriate statistical methods. Result stated that triglyceride level increased by 21.35 mg/dl (male) and 17.11 mg/dl (female) in control group. Same time triglyceride level found to be reduced significantly in treatment group by 11.51 mg/dl (male) and 16.7 mg/dl (female). Serum cholesterol level in control group found to be increased by 12.6 mg/dl (male) and 8.66 mg/dl (female), but serum cholesterol decreased by 14.8 mg/dl (male) and 13 mg/dl (female) in HIV patients receiving Spirulina supplementation.

Initiation of nutritional support is delayed in critically ill obese patients: a multicenter cohort study , ,

A high catabolic rate characterizes the acute phase of critical illness. Guidelines recommend an early nutritional support, regardless of the previous nutritional status. We aimed to assess whether the nutritional status of patients, which was defined by the body mass index (BMI) at admission in an intensive care unit (ICU), affected the time of nutritional support initiation. We conducted a cohort study that reported a retrospective analysis of a multicenter ICU database (OUTCOMEREA) by using data prospectively entered from January 1997 to October 2012. Patients who needed orotracheal intubation within the first 72 h and >3 d were included. Data from 3257 ICU stays were analyzed. The delay before feeding was different according to BMI groups (P = 0.035). The delay was longer in obese patients [BMI (in kg/m²) ≥30; n = 663] than in other patients with either low weight (BMI <20; n = 501), normal weight (BMI ≥20 and <25; n = 1135), or overweight (BMI ≥25 and <30; n = 958). The association between nutritional status and a delay in nutrition initiation was independent of potential confounding factors such as age, sex, and diabetes or other chronic diseases. In comparison with normal weight, the adjusted RR (95% CI) associated with a delayed nutrition initiation was 0.92 (0.86, 0.98) for patients with low weight, 1.00 (0.94, 1.05) for overweight patients, and 1.06 (1.00, 1.12) for obese patients (P = 0.004). The initiation of nutritional support was delayed in obese ICU patients. Randomized controlled trials that address consequences of early compared with delayed beginnings of nutritional support in critically ill obese patients are needed.

Exploration and comparison of inborn capacity of aerobic and anaerobic metabolisms of Saccharomyces cerevisiae for microbial electrical current production

Saccharomyces cerevisiae possesses numerous advantageous biological features, such as being robust, easily handled, mostly non-pathogenic and having high catabolic rates, etc., which can be considered as merits for being used as a promising biocatalyst in microbial fuel cells (MFCs) for electricity generation. Previous studies have developed efficient MFC configurations to convert metabolic electron shuttles, such as cytoplasmic NADH, into usable electric current. However, no studies have elucidated the maximum potential of S. cerevisiae for current output and the underlying metabolic pathways, resulting from the interaction of thousands of reactions inside the cell during MFC operation. To address these two key issues, this study used in silico metabolic engineering techniques, flux balance analysis (FBA), and flux variability analysis with target flux minimization (FATMIN), to model the metabolic perturbation of S. cerevisiae under the MFC-energy extraction. The FBA results showed that, in the cytoplasmic NADH-dependent mediated electron transfer (MET) mode, S. cerevisiae had a potential to produce currents at up to 5.781 A/gDW for the anaerobic and 6.193 A/gDW for the aerobic environments. The FATMIN results showed that the aerobic and anaerobic metabolisms are resilient, relying on six and five contributing reactions respectively for high current production. Two reactions, catalyzed by glutamate dehydrogenase (NAD) (EC 1.4.1.3) and methylene tetrahydrofolate dehydrogenase (NAD) (EC 1.5.1.5), were shared in both current-production modes and contributed to over 80% of the identified maximum current outputs. It is also shown that the NADH regeneration was much less energy costly than biomass production rate. Taken together, our finding suggests that S. cerevisiae should receive more research effort for MFC electricity production.

Characterisation of yeast microbial fuel cell with the yeast Arxula adeninivorans as the biocatalyst

Yeast microbial fuel cells have received little attention to date. Yeast should be ideal MFC catalyst because they are robust, easily handled, mostly non-pathogenic organisms with high catabolic rates and in some cases a broad substrate spectrum. Here we show that the non-conventional yeast Arxula adeninvorans transfers electrons to an electrode through the secretion of a reduced molecule that is not detectable when washed cells are first resuspended but which accumulates rapidly in the extracellular environment. It is a single molecule that accumulates to a significant concentration. The occurrence of mediatorless electron transfer was first established in a conventional microbial fuel cell and that phenomenon was further investigated by a number of techniques. Cyclic voltammetry (CV) on a yeast pellet shows a single peak at 450 mV, a scan rate study showed that the peak was due to a solution species. CVs of the supernatant confirmed a solution species. It appears that, given its other attributes, A. adeninivorans is a good candidate for further investigation as a MFC catalyst.

Novel aspects of PPARα-mediated regulation of lipid and xenobiotic metabolism revealed through a nutrigenomic study

Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a major transcriptional regulator of lipid metabolism. It is activated by diverse chemicals such as fatty acids (FAs) and regulates the expression of numerous genes in organs displaying high FA catabolic rates, including the liver. The role of this nuclear receptor as a sensor of whole dietary fat intake has been inferred, mostly from high-fat diet studies. To delineate its function under low fat intake conditions (4.8% w/w), we studied the effects of five regimens with contrasted FA compositions on liver lipids and hepatic gene expression in wild-type and PPARalpha-deficient mice. Diets containing polyunsaturated FAs reduced hepatic fat stores in wild-type mice. Only sunflower, linseed, and fish oil diets lowered hepatic lipid stores in PPARalpha-/- mice, a model of progressive hepatic triglyceride accumulation. These beneficial effects were associated, in particular, with dietary regulation of Delta9-desaturase in both genotypes, and with a newly identified PPARalpha-dependent regulation of lipin. Furthermore, hepatic levels of 18-carbon essential FAs (C18:2omega6 and C18:3omega3) were elevated in PPARalpha-/- mice, possibly due to the observed reduction in expression of the Delta6-desaturase and of enoyl-coenzyme A isomerases. Effects of diet and genotype were also observed on the xenobiotic metabolism-related genes Cyp3a11 and CAR. Together, our results suggest that dietary FAs represent--even under low fat intake conditions--a beneficial strategy to reduce hepatic steatosis. Under such conditions, we established the role of PPARalpha as a dietary FA sensor and highlighted its importance in regulating hepatic FA content and composition.

Glucose Metabolism in Cancer: IMPORTANCE OF TRANSCRIPTION FACTOR-DNA INTERACTIONS WITHIN A SHORT SEGMENT OF THE PROXIMAL REGION OF THE TYPE II HEXOKINASE PROMOTER

A common signature of many cancers is a high glucose catabolic rate frequently dependent on the overexpression of Type II hexokinase (HKII), a mitochondrial bound enzyme that also suppresses cell death. As the tumor HKII promoter plays a significant role in HKII overexpression, studies reported here were undertaken to identify both the major regions and transcription factors involved under tumor-like conditions. Reporter gene assays following transfection of hepatoma cells with decreasing segments of the HKII promoter traced its known strength to the proximal region (-281 to -35). Mutational analyses showed that in this short region GC boxes 1, 2, 5, and 6, a CCAAT box, an inverted CCAAT box, and CRE are involved in promoter activation. Other studies demonstrated binding of transcription factors Sp1, Sp2, and Sp3 to GC boxes 1 and 6, Sp1 and Sp2 to GC boxes 2 and 5, NF-Y to CCAAT boxes, and CREB, ATF1, and CREM to CRE. In addition, transfection studies involving Sp1, Sp2, Sp3, CREB, and NFY (dominant negative form) provided evidence that these proteins are promoter activators. Finally, alignment of available HK proximal promoters showed strong conservation only among HKII sequences. These findings implicate signaling pathways directed to a short segment of the proximal region of the HKII promoter as major contributors to HKII overexpression in many cancers.

Expansion dynamics, metabolite composition and substance transfer of the primary root growth zone ofZea maysL. grown in different external nutrient availabilities

ABSTRACTA combined analysis of growth and metabolite composition was performed in primary roots ofZea maysL. (Var. Alexander). The seedlings were hydroponically cultivated either in pure water or in complete nutrient solution. The overall root growth performance was similar in both treatments. Yet, digital image sequence processing methods resolved, that growth distribution and oscillatory movements within the growth zone depended strongly on external nutrient availability. Metabolite concentration profiles were similar in both treatments for most investigated metabolites, indicating a thorough mobilization of nutrient resources from the seed, but concentrations of glutamine, glutamic acid, NO3–, NH4+, malate and citrate showed pronounced differences between treatments. No diurnal variations in metabolite concentrations were found. Deposition rate profiles were in general more similar to relative elemental growth rate profiles than concentration profiles and were not affected by the treatment. Major ions were deposited maximally in front of the centre of growth activity, while greatest hexose deposition was found behind that. Relative to their abundance in the root growth zone, net rates of transfer from mature tissue were highest for sucrose, glutamic acid and aspartic acid, whereas glucose, fructose and most amino acids inversely showed high net rates of transfer out of the root growth zone, indicating a high catabolic rate for those substances there. NO3–, but not other nutrients, was transferred to a great extent from the root growth zone to the mature tissue in nutrient solution. Overall, the results show, that a careful analysis of growth dynamics allows quantifying and interpreting a number of important flux parameters in the growing organ and that the performance of the primary root does not depend strongly on external nutrient availability.

Carbon and electron flow in Clostridium cellulolyticum grown in chemostat culture on synthetic medium.

Previous results indicated poor sugar consumption and early inhibition of metabolism and growth when Clostridium cellulolyticum was cultured on medium containing cellobiose and yeast extract. Changing from complex medium to a synthetic medium had a strong effect on (i) the specific cellobiose consumption, which was increased threefold; and (ii) the electron flow, since the NADH/NAD+ ratios ranged from 0.29 to 2.08 on synthetic medium whereas ratios as high as 42 to 57 on complex medium were observed. These data indicate a better control of the carbon flow on mineral salts medium than on complex medium. By continuous culture, it was shown that the electron flow from glycolysis was balanced by the production of hydrogen gas, ethanol, and lactate. At low levels of carbon flow, pyruvate was preferentially cleaved to acetate and ethanol, enabling the bacteria to maximize ATP formation. A high catabolic rate led to pyruvate overflow and to increased ethanol and lactate production. In vitro, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and ethanol dehydrogenase levels were higher under conditions giving higher in vivo specific production rates. Redox balance is essentially maintained by NADH-ferredoxin reductase-hydrogenase at low levels of carbon flow and by ethanol dehydrogenase and lactate dehydrogenase at high levels of carbon flow. The same maximum growth rate (0.150 h-1) was found in both mineral salts and complex media, proving that the uptake of nutrients or the generation of biosynthetic precursors occurred faster than their utilization. On synthetic medium, cellobiose carbon was converted into cell mass and catabolized to produce ATP, while on complex medium, it served mainly as an energy supply and, if present in excess, led to an accumulation of intracellular metabolites as demonstrated for NADH. Cells grown on synthetic medium and at high levels of carbon flow were able to induce regulatory responses such as the production of ethanol and lactate dehydrogenase.

Aberrant glycolytic metabolism of cancer cells: a remarkable coordination of genetic, transcriptional, post-translational, and mutational events that lead to a critical role for type II hexokinase.

For more than two-thirds of this century we have known that one of the most common and profound phenotypes of cancer cells is their propensity to utilize and catabolize glucose at high rates. This common biochemical signature of many cancers, particularly those that are poorly differentiated and proliferate rapidly, has remained until recently a "metabolic enigma." However, with many advances in the biological sciences having been applied to this problem, cancer cells have begun to reveal their molecular strategies in maintaining an aberrant metabolic behavior. Specifically, studies performed over the past two decades in our laboratory demonstrate that hexokinase, particularly the Type II isoform, plays a critical role in initiating and maintaining the high glucose catabolic rates of rapidly growing tumors. This enzyme converts the incoming glucose to glucose-6-phosphate, the initial phosphorylated intermediate of the glycolytic pathway and an important precursor of many cellular "building blocks." At the genetic level the tumor cell adapts metabolically by first increasing the gene copy number of Type II hexokinase. The enzyme's gene promoter, in turn, shows a wide promiscuity toward the signal transduction cascades active within tumor cells. It is activated by glucose, insulin, low oxygen "hypoxic" conditions, and phorbol esters, all of which enhance the rate of transcription. Also, the tumor cell uses the tumor suppressor p53, which is usually modified by mutations to debilitate cell cycle controls, to further activate hexokinase gene transcription. This results in both enhanced levels of the enzyme, which binds to mitochondrial porins thus gaining preferential access to mitochondrially generated ATP, and in a decreased susceptibility to product inhibition and proteolytic degradation. Significantly, these multiple strategies all work together to enable tumor cells to develop a metabolic strategy compatible with rapid proliferation and prolonged survival.

Impact of the nutritional regimen on protein catabolism and nitrogen balance in patients with acute renal failure.

Patients with acute renal failure are in substantial negative nitrogen balance as a result of their extremely high protein catabolic rates. We prospectively evaluated a series of patients with acute renal failure managed with continuous venovenous hemofiltration to determine which nutritional and nonnutritional variables might influence protein catabolism and nitrogen balance. Forty consecutive patients (aged 52 +/- 20 years; mean +/- SD) were monitored for 357 treatment days (average treatment duration 8.9 +/- 8.6 days). All data (including nutritional regimen, laboratory values, APACHE II score, administered blood products, hemofiltration parameters, and medications) were collected daily. For all patients, the mean normalized protein catabolic rate was 1.4 +/- 0.5 g/kg per day. The rate did not differ between those who received nutrition support and those who did not. The net nitrogen deficit was less in those patients receiving nutrition support (-6.0 +/- 5.2 vs -14.0 +/- 5.6 g N/d; p = .02). Using regression techniques (adjusted for the within-person correlation and the previous day's normalized protein catabolic rate), the level of protein and energy provision and the interaction between protein and energy provision were predictive of the normalized protein catabolic rate. Predicted values, using this equation, suggest that at low protein administration rates (< 1 g/kg per day), increasing energy provision may reduce the protein catabolism. However, at this level of protein provision, patients remain in negative nitrogen balance. At protein administration rates necessary to achieve nitrogen balance (approximately 1.5 to 1.8 g/kg per day), protein catabolism may increase. Providing relatively low levels of energy may diminish the magnitude of this increase. These results suggest that the optimal nutritional regimen for patients with acute renal failure may require a high-protein (approximately 1.5 to 1.8 g/kg per day) and a relatively low-energy (approximately 25 to 35 kcal/kg per day) content.

Glucose metabolism in cancer cells: immunogold localization of hexokinase to the outer mitochondrial membrane

Hexokinase (ATP: D-hexose 6-phophotransferase EC 2.7.1.1) is the first enzyme of the glycolytic pathway which commits glucose to catabolism by catalyzing the phosphorylation of glucose with ATP. Previous studies have shown diat hexokinase activity is markedly elevated in rapidly growing tumor cells exhibiting high glucose catabolic rates. A large fraction (50-80%) of this enzyme activity is bound to the mitochondrial fraction (1,2) where it has preferred access to ATP (3). In contrast,the hexokinase activity of normal tissues is quite low, with one exception being brain which is a glucose-utilizing tissue (4). Biochemical evidence involving rigorous subfractionation studies have revealed striking differences between the subcellular distribution of hexokinase in normal and tumor cells [See review by Aroraet al(4)].In the present report, we have utilized immunogold labeling techniques to evaluate die subcellular localization of hexokinase in highly glycolytic AS-30D hepatoma cells and in the tissue of its origin, i.e., rat liver.

Metabolism of low density lipoproteins in nephrotic dyslipidemia: Comparison of hypercholesterolemia alone and combined hyperlipidemia

High levels of low-density lipoprotein cholesterol (LDL) (hypercholesterolemia) are commonly present in the nephrotic syndrome. Another pattern of dyslipidemia in nephrotic patients is an elevation of both cholesterol and triglyceride levels (combined hyperlipidemia). It has been postulated that the underlying cause of nephrotic dyslipidemia is an hepatic overproduction of apolipoprotein B (apo B)-containing lipoproteins. To examine this hypothesis, the metabolism of LDL-apo B was compared between nephrotic patients with hypercholesterolemia and with combined hyperlipidemia. Thirteen patients (7 with hypercholesterolemia, and 6 with combined hyperlipidemia) underwent measurements of turnover rates of autologous LDL apo B. The results were compared to normolipidemic controls and to patients with primary combined hyperlipidemia previously studied in our laboratory. Nephrotic patients with hypercholesterolemia generally had: (a) lower fractional catabolic rates of LDL apo B than normolipidemic healthy individuals; (b) LDL particles enriched in cholesterol; but (c) no overproduction of LDL apo B. In contrast, patients with combined hyperlipidemia were found to have: (a) high fractional catabolic rates for LDL apo B compared to normolipidemic controls; (b) cholesterol-poor LDL particles; and (c) markedly elevated production rates for LDL. Also, for the group as a whole, there was a positive correlation between plasma triglyceride levels and fractional catabolic rates. These data indicate that the metabolism of LDL is strikingly different between the two forms of nephrotic dyslipidemia. Although there may be common mechanisms contributing to LDL levels in nephrotic patients, there also appears to be a divergence of mechanisms depending on whether hypertriglyceridemia is associated with hypercholesterolemia.

Comparison of the intravascular metabolism of cholesteryl esters and apoproteins of plasma low- and high-density lipoproteins in the rat ( Rattus norvegicus), an animal species without plasma cholesteryl ester transfer activity

1. 1. The intravascular metabolism of the cholesteryl esters (CE) and apoproteins of low density lipoproteins (LDL) and high density lipoproteins (HDL) was compared in the rat, an animal species without plasma cholesteryl ester transfer activity (CETA). 2. 2. The apoproteins and the CE of LDL had identical catabolic rates, and there was no transfer of LDL CE to other lipoprotein classes. 3. 3. The CE of the HDL, however, had higher catabolic rates than the apoproteins, and there was transfer of HDL CE to LDL but not to very low density lipoproteins.

Apo C-II deficiency type Bari.

We formerly studied an Italian family with apo C-II deficiency. Two probands were homozygous for the defect (unmeasurable circulating apolipoprotein C-II and absence of C-II bands on immunoelectrophoresis). We documented the synthesis of the protein at the intestinal level in the probands with immunohistological techniques. With the purpose of investigating the molecular basis of the defect, Southern analysis, polymerase chain reaction (PCR) amplification and sequence analysis were carried out on one of the two cases. We identified a point mutation C to G transversion in the third exon of the gene causing a premature stop codon. Our hypothesis is that the truncated protein of 36 aa., instead of 79 aa., lacks its functional domain. This causes inefficiency in the activation of lipoprotein lipase (LPL) and the instability of the circulating molecule, which could have an higher catabolic rate compared to a normal protein. The faster disappearance from the circulating compartment make it unmeasurable. The mutation destroys a Rsa I site, present in the normal gene sequence. We suggest the use of this site for a rapid Restriction Fragment Length Polymorphism (RFLP) on PCR amplification products to screen this defect in the Italian population.

A form of familial hypobetalipoproteinaemia not due to a mutation in the apolipoprotein B gene

Familial hypobetalipoproteinaemia (FHBL) is a dominant disorder of lipoprotein metabolism characterized by levels of apolipoprotein B-carrying lipoproteins (VLDL, IDL and LDL) which are 50% of the normal levels in the heterozygotes and almost absent in the homozygotes. Several reports have recently shown that the underlying defect in FHBL involves different mutations in the apo B gene which lead to reduced levels of apo B mRNA or to the production of truncated forms of apo B having either a lower synthetic rate or a higher catabolic rate than normal apo B. We here present a three-generation family with several FHBL members in which the linkage analysis shows absence of co-segregation between apo B gene alleles and the hypocholesterolaemic phenotype. We conclude that a dominantly transmitted mutation in a gene other than that for apo B is responsible for the low plasma cholesterol levels.

Glucose phosphorylation in tumor cells. Cloning, sequencing, and overexpression in active form of a full-length cDNA encoding a mitochondrial bindable form of hexokinase.

In rapidly growing tumor cells exhibiting high glucose catabolic rates, the enzyme hexokinase is markedly elevated and bound in large amounts (50-80% of the total cell activity) to the outer mitochondrial membrane (Arora, K.K., and Pedersen, P.L. (1988) J. Biol. Chem. 263, 17422-17428; Parry, D.M., and Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912). In extending these studies, we have isolated a cDNA clone of hexokinase from a lambda gt11 library of the highly glycolytic, c37 mouse hepatoma cell line. This clone, comprising 4,198 base pairs, contains a single open reading frame of 2,754 nucleotides which encode a 918-amino acid hexokinase with a mass of 102,272 daltons. This enzyme exhibits, respectively, 68 and 32 amino acid differences, including several charge differences, from the recently sequenced human kidney and rat brain enzymes. The putative glucose and ATP binding domains present in the latter two enzymes and in rat liver glucokinase are conserved in the tumor enzyme. At its N-terminal region, tumor hexokinase has a 12-amino acid hydrophobic stretch which is present in the rat brain enzyme but absent in the rat liver glucokinase, a cytoplasmic enzyme. The mature tumor hexokinase protein has been overexpressed in active form in Escherichia coli and purified 9-fold. The overexpressed enzyme binds to rat liver mitochondria in the presence of MgCl2. This is the first report describing the cloning and sequencing of a tumor hexokinase, and the first report documenting the overexpression of any hexokinase type in E. coli. Questions pertinent to the enzyme's mechanism, regulation, binding to mitochondria, and its marked elevation in tumor cells can now be addressed.

CONTINUOUS ARTERIOVENOUS HAEMODIALYSIS IN CRITICALLY ILL PATIENTS

Continuous arteriovenous haemodialysis (CAVHD) is a new treatment for critically ill patients with renal failure that combines convective and diffusive solute removal. The clearance of urea (14·8-22·1 ml/min) is sufficient to achieve a steady-state urea concentration of 22 mmol/1, even in patients with a high catabolic rate and cardiovascular instability. The technique is simple and does not involve the use of blood pumps or specialised staff. Initial experience in 36 critically ill patients with renal failure indicates that it is safe and reliable, with less associated morbidity than other techniques.

Prevention of prednisone-induced negative nitrogen balance. Effect of dietary modification on urea generation rate in patients on hemodialysis receiving high-dose glucocorticoids.

To assess whether increasing dietary protein and calorie intake can ameliorate the negative nitrogen balance induced by 70 to 120 mg/d prednisone, we studied nitrogen intake and net urea generation rate in patients undergoing hemodialysis for 10 to 14 days after renal transplantation. Seven patients receiving prednisone with moderately restricted protein (0.73 +/- 0.03 g/kg of body weight per day) and calorie (20 +/- 4 kcal/kg of body weight per day) intake had high urea nitrogen generation rates (199 +/- 18 mg/kg.d) and protein catabolic rates (1.45 +/- 0.12 g/kg.d) and were in marked negative protein balance (-0.72 +/- 0.12 g/kg.d). An increase in protein (1.30 +/- 0.06 g/kg.d) and calorie (33 +/- 3 kcal/kg.d) consumption in another eight prednisone-treated patients resulted in protein balance (-0.02 +/- 0.12 g/kg.d) without further increasing urea generation (174 +/- 9 mg/kg.d). Six control patients undergoing hemodialysis after surgery who were not receiving prednisone had lower urea generation rates (109 +/- 15 mg/kg.d) and were in nitrogen balance. Nitrogen wasting is therefore not an inevitable consequence of high-dose glucocorticoid therapy and can be effectively prevented by simple nutritional modification without increasing hemodialytic requirements.