Methods for Quantitative Creatinine Determination.

Reliable measurement of creatinine is necessary to assess kidney function, and also to quantitate drug levels and diagnostic compounds in urine samples. The most commonly used methods are based on the Jaffe principal of alkaline creatinine-picric acid complex color formation. However, other compounds commonly found in serum and urine may interfere with Jaffe creatinine measurements. Therefore, many laboratories have made modifications to the basic method to remove or account for these interfering substances. This appendix will summarize the basic Jaffe method, as well as a modified, automated version. Also described is a high performance liquid chromatography (HPLC) method that separates creatinine from contaminants prior to direct quantification by UV absorption. Lastly, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described that uses stable isotope dilution to reliably quantify creatinine in any sample. This last approach has been recommended by experts in the field as a means to standardize all quantitative creatinine methods against an accepted reference. © 2017 by John Wiley & Sons, Inc.

An early record of Meloidogyne fallax from Ireland.

Root-knot nematodes, Meloidogyne spp., cause huge economic losses worldwide. Currently, three Meloidogyne spp. are present on the quarantine A2 list of EPPO, Meloidogyne chitwoodi, Meloidogyne fallax and Meloidogyne enterolobii. As a quarantine organism, Meloidogyne fallax has been detected in England and Northern Ireland on sport turf in 2011, and in England on leek in 2013. However, its presence in Ireland has probably been overlooked since 1965, when Mr. John F. Moore and Dr. Mary T. Franklin had detected a new Meloidogyne species for that time. While the relevant data was recorded and a preliminary manuscript describing the species was prepared but never submitted for publication, and together with the original slides, pictures and drawings, it was restudied recently. We compared the population of Irish Meloidogyne sp. to other similar Meloidogyne spp. Careful observation and comparison shows that it belongs to Meloidogyne fallax. The characters found to be common for Irish Meloidogyne sp. and Meloidogyne fallax are female stylet length (14.6 µm) with oval to rounded basal knobs, oval shaped perineal pattern with moderately high dorsal arch, slender stylet in males (18.5 µm) with set off and rounded basal knobs, slightly set off male head with one post-labial annule and incomplete transverse incisures, and second-stage juveniles with large and rounded stylet basal knobs, and a gradually tapering tail (46.9 µm) with a broadly rounded tip and a clearly delimitated smooth hyaline part sometimes marked by constrictions (12.9 µm). The host test and gall formation also correspond to Meloidogyne fallax. The identification could not be additionally supported by molecular analysis, as we were unable to extract DNA from the old permanent slides. Nevertheless, our study reveals that the Meloidogyne species detected in Ireland in 1965 belongs to Meloidogyne fallax.

Carnitine Palmitoyltransferase 1b Deficiency Protects Mice from Diet-Induced Insulin Resistance.

BACKGROUND: Carnitine Palmitoyl Transferase 1 (CPT1) is the rate-limiting enzyme governing long-chain fatty acid entry into mitochondria. CPT1 inhibitors have been developed and exhibited beneficial effects against type II diabetes in short-term preclinical animal studies. However, the long-term effects of treatment remain unclear and potential non-specific effects of these CPT1 inhibitors hamper in-depth understanding of the potential molecular mechanisms involved. METHODS: We investigated the effects of restricting the activity of the muscle isoform CPT1b in mice using heterozygous CPT1b deficient (Cpt1b+/-) and Wild Type (WT) mice fed with a High Fat Diet (HFD) for 22 weeks. Insulin sensitivity was assessed using Glucose Tolerance Test (GTT), insulin tolerance test and hyperinsulinemic euglycemic clamps. We also examined body weight/composition, tissue and systemic metabolism/energetic status, lipid profile, transcript analysis, and changes in insulin signaling pathways. RESULTS: We found that Cpt1b+/- mice were protected from HFD-induced insulin resistance compared to WT littermates. Cpt1b+/- mice exhibited elevated whole body glucose disposal rate and skeletal muscle glucose uptake. Furthermore, Cpt1b+/- skeletal muscle showed diminished ex vivo palmitate oxidative capacity by ~40% and augmented glucose oxidation capacity by ~50% without overt change in whole body energy metabolism. HFD feeding Cpt1b+/- but not WT mice exhibited well-maintained insulin signaling in skeletal muscle, heart, and liver. CONCLUSION: The present study on a genetic model of CPT1b restriction supports the concept that partial CPT1b inhibition is a potential therapeutic strategy.

Comparison of methods, storage conditions, and time to analysis of serum and urine creatinine measured from microsamples by liquid chromatography mass spectrometery (LC/MS) vs. Jaffe.

BACKGROUND: Measurement of serum creatinine (SCr) and urine creatinine (UCr) is regularly used in clinical and research settings. For small animal experiments and for studies in which sample collection is spare (i.e. neonatal cohorts), measuring SCr and UCr using tiny amounts of sample (as low as 10 mcl) would maximize exploration and minimize iatrogenic blood loss. METHODS: We performed an evaluation in six healthy adults to determine differences between SCr and UCr values in different methodologies and storage environments and time. Study was conducted using 20 mcl of sample. Analyses were done using two-way repeated measures of ANOVA. RESULTS: Scr values showed no significant differences between LC/MS vs. Jaffe. However, the SCr using LC/MS method was lowest when measured immediately compared to other time points (F = 7.2; P< 0.001). Similarly, Jaffe measurements showed changes in the mean differences over time; however, these were not significant. UCr values were consistently higher using LC/MS than Jaffe (F = 19; P< 0.01), and UCr changed over time (F = 8.7; P < 0.02). In addition, the interaction term for method and time was also significant (F = 5.8; P < 0.04) which reflects the stability of the Jaffe measurements over time whereas the LC/MS measurements declined; especially after being frozen for 1 year (P < 0.001). CONCLUSION: UCr measured by Jaffe is lower than samples measured by LC/MS. UCr measurements by LC/MS vary more over time, mostly due to the sample measured after 1 year; therefore, storage of urine for more than 90 days measured by LC/MS may provide altered results.

Carnitine Palmitoyltransferase 1b Deficient Mice Develop Severe Insulin Resistance After Prolonged High Fat Diet Feeding.

BACKGROUND: Carnitine palmitoyltransferase 1 (CPT1) is the rate-limiting enzyme governing the entry of long-chain acyl-CoAs into mitochondria. Treatments with CPT1 inhibitors protect against insulin resistance in short-term preclinical animal studies. We recently reported that mice with muscle isoform CPT1b deficiency demonstrated improved insulin sensitivity when fed a High Fat-Diet (HFD) for up to 5 months. In this follow up study, we further investigated whether the insulin sensitizing effects of partial CPT1b deficiency could be maintained under a prolonged HFD feeding condition. METHODS: We investigated the effects of CPT1b deficiency on HFD-induced insulin resistance using heterozygous CPT1b deficient (Cpt1b+/-) mice compared with Wild Type (WT) mice fed a HFD for a prolonged period of time (7 months). We assessed insulin sensitivity using hyperinsulinemic-euglycemic clamps. We also examined body composition, skeletal muscle lipid profile, and changes in the insulin signaling pathways of skeletal muscle, liver, and adipose tissue. RESULTS: We found that Cpt1b+/- mice became severely insulin resistant after 7 months of HFD feeding. Cpt1b+/- mice exhibited a substantially reduced glucose infusion rate and skeletal muscle glucose uptake. While Cpt1b+/- mice maintained a slower weight gain with less fat mass than WT mice, accumulation of lipid intermediates became evident in the muscle of Cpt1b+/- but not WT mice after 7 months of HFD feeding. Insulin signaling was impaired in the Cpt1b+/- as compared to the WT muscles. CONCLUSION: Partial CPT1b deficiency, mimicking CPT1b inhibition, may lead to impaired insulin signaling and insulin sensitivity under a prolonged HFD feeding condition. Therefore, further studies on the potential detrimental effects of prolonged therapy with CPT1 inhibition are necessary in the development of this potential therapeutic strategy.