Validation of a FFQ to estimate the intake of PUFA using plasma phospholipid fatty acids and weighed foods records

Due to the growing knowledge about the role of specific fatty acids in health and disease, dietary intake measurements of individual fatty acids or classes of fatty acids are becoming increasingly important. The objective of this study was to evaluate the ability of the Nambour FFQ to estimate intakes of specific fatty acids, particularly PUFA. The study population was a sub-sample of adult participants in a randomised controlled trial of [beta]-carotene and sunscreen in the prevention of skin cancer (n 43). Dietary intake was assessed by a self-administered FFQ and a weighed food record (WFR). Non-fasting blood samples were collected and analysed for plasma phospholipid fatty acids. Median intakes on the FFQ were generally higher than the WFR except for the n-3 PUFA groups, where the FFQ estimated higher intakes. Correlations between the FFQ and WFR were moderate (r 0–32-0-59) except for trans fatty acids (r 0–03). Correlations between each of the dietary assessment methods and the plasma phospholipids were poor for all fatty acids other than the PUFA. Using the methods of triads approach, the FFQ validity coefficients for total n-3 fatty acids, total long chain n-3 fatty acids, EPA, arachidonic acid, docosapentaenoic acid and DHA were 0–50, 0–63, 0–45 and 0–62 and 0–62, respectively. For most fatty acids, the FFQ adequately estimates group mean fatty acid intakes and can adequately rank individuals; however, the ability of this FFQ to estimate trans fatty acids was poor.

Association of television viewing with fasting and 2-h postchallenge plasma glucose levels in adults without diagnosed diabetes

OBJECTIVE—We examined the associations of television viewing time with fasting plasma glucose (FPG) and 2-h postchallenge plasma glucose (2-h PG) levels in Australian adults.

RESEARCH DESIGN AND METHODS—A total of 8,357 adults aged >35 years who were free from diagnosed diabetes and who attended a population-based cross-sectional study (Australian Diabetes, Obesity and Lifestyle Study [AusDiab]) were evaluated. Measures of FPG and 2-h PG were obtained from an oral glucose tolerance test. Self-reported television viewing time (in the previous week) was assessed using an interviewer-administered questionnaire. Homeostasis model assessment (HOMA) of insulin sensitivity (HOMA-%S) and ß-cell function (HOMA-%B) were calculated based on fasting glucose and insulin concentrations.

RESULTS—After adjustment for confounders and physical activity time, time spent watching television in women was positively associated with 2-h PG, log fasting insulin, and log HOMA-%B and inversely associated with log HOMA-%S (P < 0.05) but not with FPG. No significant associations were observed with glycemic measures in men. The ß-coefficients across categories of average hours spent watching television per day (<1.0, 1.0–1.9, 2.0–2.9, 3.0–3.9, and ≥4.0) for 2-h PG in women were 0 (reference), 0.009, 0.047, 0.473, and 0.501, respectively (P for trend = 0.02).

CONCLUSIONS—Our findings highlight the unique deleterious relationship of sedentary behavior (indicated by television viewing time) and glycemic measures independent of physical activity time and adiposity status. These relationships differed according to sex and type of glucose measurement, with the 2-h PG measure being more strongly associated with television viewing. The findings suggest an important role for reducing sedentary behavior in the prevention of type 2 diabetes and cardiovascular disease, especially in women.

Objectively measured light-intensity physical activity is independently associated with 2-H plasma glucose

Objective: We examined the associations of objectively measured sedentary time, light-intensity physical activity, and moderate- to vigorous-intensity activity with fasting and 2-h postchallenge plasma glucose in Australian adults.

Research Design and Methods: A total of 67 men and 106 women (mean age ± SD 53.3 ± 11.9 years) without diagnosed diabetes were recruited from the 2004–2005 Australian Diabetes, Obesity, and Lifestyle (AusDiab) study. Physical activity was measured by Actigraph  accelerometers worn during waking hours for 7 consecutive days and summarized as sedentary time (accelerometer counts/min <100; average hours/day), light-intensity (counts/min 100-1951), and moderate- to vigorous-intensity (counts/min ≥1,952). An oral glucose tolerance test was used to ascertain 2-h plasma glucose and fasting plasma glucose.

Results: After adjustment for confounders (including waist circumference), sedentary time was positively associated with 2-h plasma glucose (b = 0.29, 95% CI 0.11–0.48, P = 0.002); light-intensity activity time (b = –0.25, –0.45 to –0.06, P = 0.012) and moderate- to vigorous-intensity activity time (b = –1.07, –1.77 to –0.37, P = 0.003) were negatively associated. Light-intensity activity remained significantly associated with 2-h plasma glucose following further adjustment for moderate- to vigorous-intensity activity (b = –0.22, –0.42 to –0.03, P = 0.023). Associations of all activity measures with fasting plasma glucose were nonsignificant (P > 0.05).

Conclusions: These data provide the first objective evidence that light-intensity physical activity is beneficially associated with blood glucose and that sedentary time is unfavorably associated with blood glucose. These objective data support previous findings from studies using self-report measures, and suggest that substituting light-intensity activity for television viewing or other sedentary time may be a practical and achievable preventive strategy to reduce the risk of type 2 diabetes and cardiovascular disease.

Plasma phospholipid and dietary fatty acids as predictors of type 2 diabetes: interpreting the role of linoleic acid 1-3

Background: Dietary fatty acids may be associated with diabetes but are difficult to measure accurately.

Objective: We aimed to investigate the associations of fatty acids in plasma and diet with diabetes incidence.

Design: This was a prospective case-cohort study of 3737 adults aged 36-72 y. Fatty acid intake (/kJ) and plasma phospholipid fatty acids (%) were measured at baseline, and diabetes incidence was assessed by self-report 4 y later. Logistic regression excluding (model 1) and including (model 2) body mass index and waist-hip ratio was used to calculate odds ratios (ORs) for plasma phospholipid and dietary fatty acids.

Results: In plasma phospholipid, positive associations with diabetes were seen for stearic acid [OR model 1, highest versus lowest quintile: 4.14 (95% CI: 2.65, 6.49), P for trend < 0.0001] and total saturated fatty acids [OR model 1: 3.76 (2.43, 5.81), P for trend < 0.0001], whereas an inverse association was seen for linoleic acid [OR model 1: 0.22 (0.14, 0.36), P for trend < 0.0001]. Dietary linoleic [OR model 1: 1.77 (1.19, 2.64), P for trend = 0.002], palmitic [OR model 1: 1.65 (1.12, 2.43), P for trend = 0.012], and stearic [OR model 1: 1.46 (1.00, 2.14), P for trend = 0.030] acids were positively associated with diabetes incidence before adjustment for body size. Within each quintile of linoleic acid intake, cases had lower baseline plasma phospholipid linoleic acid proportions than did controls.

Conclusions: Dietary saturated fat intake is inversely associated with diabetes risk. More research is required to determine whether linoleic acid is an appropriate dietary substitute.

Plasma phospholipid fatty acid composition as a biomarker of habitual dietary fat intake in an ethnically diverse cohort

Background and aim
As an evaluation of fatty acid intake measurement, our aim was to examine associations between diet and plasma phospholipid (PL) fatty acids, and whether these were modified by age, sex, country of birth, fasting status, use of cholesterol-lowering medication, body size, chronic disease and other lifestyle factors.

Methods and results
Cross-sectional analysis of plasma PL fatty acid composition and dietary fatty acid intake over 12 months from a 121-item food frequency questionnaire (FFQ) in 4439 men and women aged 40–69 years, born in Australia, Greece or Italy. Crude correlation coefficients ranged from 0.18 to 0.40; and corrected correlation coefficients from 0.38 to 0.78 for total monounsaturated, polyunsaturated, n-6, n-3 fatty acids, oleic acid, linoleic acid, EPA and DHA. Weaker associations were observed for other fatty acids. The associations did not vary significantly by fasting status, use of lipid lowering medication or alcohol intake, but for some fatty acids did vary by sex, age, body mass index, country of birth, smoking and previous heart attack or diabetes.

Conclusions
The FFQ provides useful information on intakes of mono- and polyunsaturated fatty acids. Correlations did not differ by fasting status, or use of lipid-lowering medication.

Creatine supplementation increases glycogen storage but not GLUT-4 expression in human skeletal muscle

It has been speculated that creatine supplementation affects muscle glucose metabolism in humans by increasing muscle glycogen storage and up-regulating GLUT-4 protein expression. In the present study, we assessed the effects of creatine loading and prolonged supplementation on muscle glycogen storage and GLUT-4 mRNA and protein content in humans. A total of 20 subjects participated in a 6-week supplementation period during which creatine or a placebo was ingested. Muscle biopsies were taken before and after 5 days of creatine loading (20 g.day(-1)) and after 6 weeks of continued supplementation (2 g.day(-1)). Fasting plasma insulin concentrations, muscle creatine, glycogen and GLUT-4 protein content as well as GLUT-4, glycogen synthase-1 (GS-1) and glycogenin-1 (Gln-1) mRNA expression were determined. Creatine loading significantly increased total creatine, free creatine and creatine phosphate content with a concomitant 18 +/- 5% increase in muscle glycogen content (P<0.05). The subsequent use of a 2 g.day(-1) maintenance dose for 37 days did not maintain total creatine, creatine phosphate and glycogen content at the elevated levels. The initial increase in muscle glycogen accumulation could not be explained by an increase in fasting plasma insulin concentration, muscle GLUT-4 mRNA and/or protein content. In addition, neither muscle GS-1 nor Gln-1 mRNA expression was affected. We conclude that creatine ingestion itself stimulates muscle glycogen storage, but does not affect muscle GLUT-4 expression.

Creatine transporters: a reappraisal

Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na⁺/Cl^--dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to severe retardation of speech and mental development, accompanied by the absence of Cr in the brain.
In order to characterize CrT protein(s) on a biochemical level, antibodies were raised against synthetic peptides derived from the N- and C-terminal cDNA sequences of the putative CrT-1 protein. In total homogenates of various tissues, both antibodies, directed against these different epitopes, recognize the same two major polypetides on Western blots with apparent Mr of 70 and 55 kDa. The C-terminal CrT antibody (α-CrTCOOH) immunologically reacts with proteins located at the inner membrane of mitochondria as determined by immuno-electron microscopy, as well as by subfractionation of mitochondria. Cr-uptake experiments with isolated mitochondria showed these organelles were able to transport Cr via a sulfhydryl-reagent-sensitive transporter that could be blocked by anti-CrT antibodies when the outer mitochondrial membrane was permeabilized. We concluded that mitochondria are able to specifically take-up Cr from the cytosol, via a low-affinity CrT, and that the above polypeptides would likely represent mitochondrial CrT(s). However, by mass spectrometry techniques, the immunologically reactive proteins, detected by our anti-CrT antibodies, were identified as E2 components of the agr-keto acid dehydrogenase multi enzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain keto acid dehydrogenase (BC-KADH) and α-ketoglutarate dehydrogenase (α-KGDH). The E2 components of PDH are membrane associated, whilst it would be expected that a mitochondrial CrT would be a transmembrane protein. Results of phase partitioning by Triton X-114, as well as washing of mitochondrial membranes at basic pH, support that these immunologically cross-reactive proteins are, as expected for E2 components, membrane associated rather than transmembrane. On the other hand, the fact that mitochondrial Cr uptake into intact mitoplast could be blocked by our α-CrTCOOH antibodies, indicate that our antisera contain antibodies reactive to proteins involved in mitochondrial transport of Cr. The presence of specific antibodies against CrT is also supported by results from plasma membrane vesicles isolated from human and rat skeletal muscle, where both 55 and 70 kDa polypeptides disappeared and a single polypeptide with an apparent electrophoretic mobility of ~ 60 kDa was enriched This latter is most likely representing the genuine plasma membrane CrT.
Due to the fact that all anti-CrT antibodies that were independently prepared by several laboratories seem to cross-react with non-CrT polypeptides, specifically with E2 components of mitochondrial dehydrogenases, further research is required to characterise on a biochemical/biophysical level the CrT polypeptides, e.g. to determine whether the ~ 60 kDa polypeptide is indeed a bona-fide CrT and to identify the mitochondrial transporter that is able to facilitate Cr-uptake into these organelles. Therefore, the anti-CrT antibodies available so far should only be used with these precautions in mind. This holds especially true for quantitation of CrT polypeptides by Western blots, e.g. when trying to answer whether CrT's are up- or down-regulated by certain experimental interventions or under pathological conditions.
In conclusion, we still hold to the scheme that besides the high-affinity and high-efficiency plasmalemma CrT there exists an additional low affinity high Km Cr uptake mechanism in mitochondria. However, the exact biochemical nature of this mitochondrial creatine transport, still remains elusive. Finally, similar to the creatine kinase (CK) isoenzymes, which are specifically located at different cellular compartments, also the substrates of CK are compartmentalized in cytosolic and mitochondrial pools. This is in line with ¹⁴C-Cr-isotope tracer studies and a number of [³¹P]-NMR magnetization transfer studies, as well as with recent [¹H]-NMR spectroscopy data.