Adam S. Ptolemy

Whey Protein Supplementation During Resistance Training Augments Lean Body Mass

Compared to soy, whey protein is higher in leucine, absorbed quicker and results in a more pronounced increase in muscle protein synthesis. Objective: To determine whether supplementation with whey promotes greater increases in muscle mass compared to soy or carbohydrate, we randomized non-resistance-trained men and women into groups who consumed daily isocaloric supplements containing carbohydrate (carb; n = 22), whey protein (whey; n = 19), or soy protein (soy; n = 22). Methods: All subjects completed a supervised, whole-body periodized resistance training program consisting of 96 workouts (∼9 months). Body composition was determined at baseline and after 3, 6, and 9 months. Plasma amino acid responses to resistance exercise followed by supplement ingestion were determined at baseline and 9 months. Results: Daily protein intake (including the supplement) for carb, whey, and soy was 1.1, 1.4, and 1.4 g·kg body mass−1, respectively. Lean body mass gains were significantly (p < 0.05) greater in whey (3.3 ± 1.5 kg) than carb (2.3 ± 1.7 kg) and soy (1.8 ± 1.6 kg). Fat mass decreased slightly but there were no differences between groups. Fasting concentrations of leucine were significantly elevated (20%) and postexercise plasma leucine increased more than 2-fold in whey. Fasting leucine concentrations were positively correlated with lean body mass responses. Conclusions: Despite consuming similar calories and protein during resistance training, daily supplementation with whey was more effective than soy protein or isocaloric carbohydrate control treatment conditions in promoting gains in lean body mass. These results highlight the importance of protein quality as an important determinant of lean body mass responses to resistance training.

What is a biomarker? Research investments and lack of clinical integration necessitate a review of biomarker terminology and validation schema.

Abstract A continual trend of annual growth can be seen within research devoted to the discovery and validation of disease biomarkers within both the natural and clinical sciences. This expansion of intellectual endeavours was quantified through database searches of (a) research grant awards provided by the various branches of the National Institutes of Health (NIH) and (b) academic publications. A search of awards presented between 1986 and 2009 revealed a total of 28,856 grants awarded by the NIH containing the term “biomarker”. The total funds for these awards in 2008 and 2009 alone were over

Quantification of theobromine and caffeine in saliva, plasma and urine via liquid chromatography–tandem mass spectrometry: A single analytical protocol applicable to cocoa intervention studies

2.5 billion. During the same respective timeframes, searches of “biomarker” and either “discovery”, “genomics”, “proteomics” or “metabolomics” yielded a total of 4,928 NIH grants whose combined funding exceeded

Acute effects of ingestion of a novel whey-derived extract on vascular endothelial function in overweight, middle-aged men and women

1.2 billion. The derived trend in NIH awards paralleled the annual expansion in “biomarker” literature. A PubMed search for the term, between 1990 and 2009, revealed a total of 441,510 published articles, with 38,457 published in 2008. These enormous investments and academic outputs however have not translated into the expected integration of new biomarkers for patient care. For example no proteomics derived biomarkers are currently being utilized in routine clinical management. This translational chasm necessitates a review of the previously proposed biomarker definitions and evaluation schema. A subsequent discussion of both the analytical and pre-analytical considerations for such research is also presented within. This required knowledge should aid scientists in their pursuit and validation of new biological markers of disease.

The Riddle of Protein Diagnostics: Future Bleak or Bright?

Targeted analyses of clinically relevant metabolites in human biofluids often require extensive sample preparation (e.g., desalting, protein removal and/or preconcentration) prior to quantitation. In this report, a single ultra-centrifugation based sample pretreatment combined with a designed liquid chromatography-tandem mass spectrometry (LC-MS/MS) protocol provides selective quantification of 3,7-dimethylxanthine (theobromine) and 1,3,7-trimethylxanthine (caffeine) in human saliva, plasma and urine samples. The optimized chromatography permitted elution of both analytes within 1.3 min of the applied gradient. Positive-mode electrospray ionization and a triple quadruple MS/MS instrument operated in multiple reaction mode were used for detection. (13)C(3) isotopically labeled caffeine was included as an internal standard to improve accuracy and precision. Implementing a 20-fold dilution of the isolated low MW biofluid fraction prior to injection effectively minimized the deleterious contributions of all three matrices to quantitation. The assay was linear over a 160-fold concentration range from 2.5 to 400 micromol L(-1) for both theobromine (average R(2) 0.9968) and caffeine (average R(2) 0.9997) respectively. Analyte peak area variations for 2.5 micromol L(-1) caffeine and theobromine in saliva, plasma and urine ranged from 5 and 10% (intra-day, N=10) to 9 and 13% (inter-day, N=25) respectively. The intra- and inter-day precision of theobromine and caffeine elution times were 3 and <1% for all biofluids and concentrations tested. Recoveries for caffeine and theobromine ranged from 114 to 118% and 99 to 105% at concentration levels of 10 and 300 micromol L(-1). This validated protocol also permitted the relative saliva, plasma and urine distribution of both theobromine and caffeine to be quantified following a cocoa intervention.

Towards an Ecology of Collective Innovation: Human Variome Project (HVP), Rare Disease Consortium for Autosomal Loci (RaDiCAL) and Data-Enabled Life Sciences Alliance (DELSA).

Whey protein intake reduces CVD risk, but little is known whether whey-derived bioactive peptides regulate vascular endothelial function (VEF). We determined the impact of a whey-derived extract (NOP-47) on VEF in individuals with an increased cardiovascular risk profile. Men and women with impaired brachial artery flow-mediated dilation (FMD) (n 21, age 55 (sem 1·3) years, BMI 27·8 (sem 0·6) kg/m2, FMD 3·7 (sem 0·4) %) completed a randomised, cross-over study to examine whether ingestion of NOP-47 (5 g) improves postprandial VEF. Brachial artery FMD, plasma amino acids, insulin, and endothelium-derived vasodilators and vasoconstrictors were measured for 2 h after ingestion of NOP-47 or placebo. Acute NOP-47 ingestion increased FMD at 30 min (4·6 (sem 0·5) %) and 120 min (5·1 (sem 0·5) %) post-ingestion (P< 0·05, time × trial interaction), and FMD responses at 120 min were significantly greater in the NOP-47 trial compared with placebo (4·3 (sem 0·5) %). Plasma amino acids increased at 30 min following NOP-47 ingestion (P< 0·05). Serum insulin increased at 15, 30 and 60 min (P< 0·001) following NOP-47 ingestion. No changes were observed between the trials for plasma NO∙ and prostacyclin metabolites or endothelin-1. Ingestion of a rapidly absorbed extract derived from whey protein improved endothelium-dependent dilation in older adults by a mechanism independent of changes in circulating vasoactive compounds. Future investigation is warranted in individuals at an increased CVD risk to further elucidate potential health benefits and the underlying mechanisms of extracts derived from whey.

Quantification of Galactose-1-Phosphate Uridyltransferase Enzyme Activity by Liquid Chromatography–Tandem Mass Spectrometry

> In theory, there is no difference between theory and practice. In practice, there is. > > —variously attributed to Andrew Tannenbaum, Jan L.A. van de Snepscheut, or Yogi Berra The theory behind an exploding kaleidoscope of protein biomarker research is that reproducible molecular differences that correlate with important medical attributes can be discovered in patient samples and that these molecules can be measured with clinical tests to obtain useful medical information. How could this not be true? Chorionic gonadotropin and cardiac troponins are very good biomarkers indeed—for pregnancy and myocardial infarction, respectively—so proteins obviously can serve as useful clinical tests. Apparently the US Food and Drug Administration (FDA) agrees, because, through 2009, it has cleared commercial clinical tests for the measurement of 109 proteins in plasma or serum. Reference laboratories offer “home brew” tests that measure 96 more, for a total of 205 proteins (10% of the proteins known to exist in plasma and 1% of the baseline human proteome) with some recognized clinical importance (1). Of course many, perhaps most, of the chronic diseases that are really difficult to manage, as well as almost all cancers, remain beyond the reach of current clinical laboratory tests. To address this need, the NIH, guided by prevailing theory and the scientific community, has devoted increasing resources to the search for biomarkers via tried-and-true research support mechanisms. Over the past 2 decades, the numbers of research grants, research dollars, and published reports self-identified as biomarker related have increased dramatically (2). Fig. 1 shows the astonishing growth in funding and publication, which we obtained with updated key-word database searches (RePORT) (3). Although we do not know how much of this growth represents real effort directed at clinical biomarkers and how much is cosmetic “repurposing” of other lines of research, the aggregate total of >

Personalized Medicine in the Age of Pharmacoproteomics: A Close up on India and Need for Social Science Engagement for Responsible Innovation in Post-Proteomic Biology

1 billion spent over the last 3 years on biomarker discovery ought to have had …

Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies

The Millennium Summit in 2000 established the Millennium Development Goals (MDGs), which were agreed upon by 193 countries and 23 international organizations to combat extreme poverty and other pressing global priorities for human development. In the December 2011 issue of CPPM, Borda-Rodriguez and Huzair present an analysis of the close ties and synergies among the MDGs, pharmacogenomics and personalized medicine. Notably, MDGs promote the creation of collective innovation, a concept with both substantive and instrumental pertinence for the personalized medicine R&D that is currently undergoing rapid globalization. The ethos for collective innovation in global health is also embodied in the Paris Declaration on Aid Effectiveness, endorsed in 2005 by more than 100 signatories, including donor and developing country governments, regional development banks and international aid agencies.

A 9-Month-Old Boy with Seizures and Discrepant Urine Tryptophan Concentrations

BACKGROUND The diagnosis of galactosemia usually involves the measurement of galactose-1-phosphate uridyltransferase (GALT) activity. Traditional radioactive and fluorescent GALT assays are nonspecific, laborious, and/or lack sufficient analytical sensitivity. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay for GALT enzyme activity measurement. METHOD Our assay used stable isotope-labeled alpha- galactose-1-phosphate ([(13)C(6)]-Gal-1-P) as an enzyme substrate. Sample cleanup and separation were achieved by reversed-phase ion-pair chromatography, and the enzymatic product, isotope-labeled uridine diphosphate galactose ([(13)C(6)]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [(13)C(6)]-Glu-1-P (265 > 79) as an internal standard. RESULTS The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) mumol x (g Hgb)(-1) x h(-1) in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 micromol x (g Hgb)(-1) x h(-1) (0.2% of normal control value). Intraassay imprecision was determined at 4 different levels (100%, 25%, 5%, and 0.2% of the normal control values), and the CVs were calculated to be 2.1%, 2.5%, 4.6%, and 9.7%, respectively (n = 3). Interassay imprecision CVs were 4.5%, 6.7%, 8.2%, and 13.2% (n = 5), respectively. The assay recoveries at the 4 levels were higher than 90%. The apparent K(m) of the 2 substrates, Gal-1-P and UDPGlc, were determined to be 0.38 mmol/L and 0.071 mmol/L, respectively. The assay in erythrocytes of 33 patients with classical galactosemia revealed no detectable activity. CONCLUSIONS This LC-MS/MS-based assay for GALT enzyme activity will be useful for the diagnosis and study of biochemically heterogeneous patients with galactosemia, especially those with uncommon genotypes and detectable but low residual activities.