Dirk M. Holstege

Diagnosis of Oleander Poisoning in Livestock

Since mid- 1989, 37 cases of oleander poisoning in livestock have been diagnosed at the California Veterinary Diagnostic Laboratory System. The most frequent source for oleander exposure was plant clippings. Sudden death was the most common presenting complaint. Other signs reported included diarrhea, pulmonary edema, tachycardia, cardiac arrhythmias, colic, and lethargy. In the past, a presumptive diagnosis of oleander poisoning could be based only on matching clinical signs with evidence of consumption of oleander. A new 2 dimensional Thin-layer chromatography analysis of ingesta for oleandrin and an awareness of lesions in heart muscle have greatly improved the ability to diagnose oleander toxicosis.

Phenolic metabolites and substantial microbiome changes in pig feces by ingesting grape seed proanthocyanidins

Proanthocyanidin (PAC) consumption has been linked to better colonic health, but PACs are poorly absorbed, making them a target for colonic metabolism. The resulting metabolites are low molecular weight and could potentially be absorbed. To understand the effects of dietary PACs it would be important to resolve the metabolic issue and link these changes to microbial population changes in a suitable model for human digestion. Here, six crossbred female pigs were fed a diet containing 1% (w/w) of MegaNatural® Gold grape seed extract (GSE) daily for 6 days. Fecal samples were analyzed by normal phase LC coupled to fluorescence detection and LC-MS/ToF. DNA was extracted from pig fecal samples and the V3/V4 region of the 16S rRNA gene was sequenced using an Illumina MiSeq. Intact parent PACs (dimer-pentamer) were observed in the feces on days 3 and 6 at similar high levels (∼400 mg kg(-1) total) during ingestion of GSE but were absent 48 h post-feeding. The major phenolic metabolites were 4-hydroxyphenylvaleric acid and 3-hydroxybenzoic acid which increased by ∼30 and 3 mg kg(-1) respectively. The GSE diet also caused an ecological shift in the microbiome, dramatically increasing Lachnospiraceae, Clostridales, Lactobacillus and Ruminococcacceae. The relationship between dietary PACs and colon health may be attributable to the altered bacterial populations or phenolic compounds in the colon.

Nicotiana Glauca Toxicosis of Cattle

rent veterinary therapy 7: small animal pratice, ed. Kirk RW, pp. 141-144. WB Saunders Co., Philadelphia, PA. 8. Miller S, Bauk TJ: 1992, Lead toxicosis in a group of cats. J Vet Diagn Invest 4:362-363. 9. Morgan RV, Moore FM, Pearce LK, Rossi T: 1991, Clinical and laboratory findings in small companion animals with lead poisoning: 347 cases (1977-1986). J Am Vet Med Assoc 199: 93-97. 10. Mount ME: 1989, Toxicology. In: Textbook of veterinary internal medicine, ed. Ettinger SJ, pp. 469-470. WB Saunders Co., Philadelphia, PA. 11. Prasse KW, Mahaffey EA: 1987, The hematopoietic system. In: Diseases of the cat: medicine and surgery, ed. Holzworth J, p. 764. WB Saunders Co., Philadelphia, PA. 12. Prescott CW: 1993, Clinical findings in dogs and cats with lead poisoning. Aust Vet J 60:270-271. 13. Puls R: 1988, Lead-cat tissue levels. In: Mineral levels in animal health: diagnostic data, ed. Puls R, p. 117. Sherpa International, Clearbrook, BC, Canada. 14. Reid FM, Oehme GW: 1989, Toxicoses. In: The cat-diseases and clinical management, ed. Sherding RG, p. 206. Churchill Livingstone, New York, NY. 15. Turner AJ, Fairbum AJ: 1979, Lead poisoning in the cat. Aust Vet Pract 9:205-207. 16. Watson ADJ: 1981, Lead poisoning in a cat. J Small Anim Pract 22:85-89. 17. Zook BC, Carpenter JL, Leeds EB: 1969, Lead poisoning in dogs. J Am Vet Med Assoc 155:1329-1342.

Toxicosis in Dairy Cattle Exposed to Poison Hemlock (Conium Maculatum) in Hay: Isolation of Conium Alkaloids in Plants, Hay, and Urine

Cattle in two herds developed signs of bloating, increased salivation and lacrimation, depression, respiratory distress, ataxia, and death after ingestion of hay that contained large amounts of poison hemlock (Conium maculatum). Twenty of 30 Angus cows and calves were affected in the first herd (2 died). In the second herd, 5 of 30 Holstein heifers were affected (1 died). The Conium alkaloids, coniine and γ -coniceine, were quantified in the hay, the plants from the responsible hayfield, and the urine of affected animals.

Vitamin Retention in Eight Fruits and Vegetables: A Comparison of Refrigerated and Frozen Storage

Four vitamins were analyzed in several fruit and vegetable commodities to evaluate the differences between fresh and frozen produce. Ascorbic acid, riboflavin, α-tocopherol, and β-carotene were evaluated in corn, carrots, broccoli, spinach, peas, green beans, strawberries, and blueberries. Samples of each commodity were harvested, processed, and analyzed for nutrient content at three storage times per treatment. Ascorbic acid showed no significant difference for five of the eight commodities and was higher in frozen samples than fresh for the remaining three commodities. Apart from broccoli and peas, which were higher and lower in frozen vs fresh samples, respectively, none of the commodities showed significant differences with respect to riboflavin content. Three commodities had higher levels of α-tocopherol in the frozen samples, while the remaining commodities showed no significant difference between fresh and frozen. β-Carotene was not found in significant amounts in blueberries, strawberries, and corn. Peas, carrots, and spinach were lower in β-carotene in the frozen samples, while green beans and spinach showed no significant difference between the two storage methods. Overall, the vitamin content of the frozen commodities was comparable to and occasionally higher than that of their fresh counterparts. β-Carotene, however, was found to decrease drastically in some commodities.

A rapid method to determine sterol, erythrodiol, and uvaol concentrations in olive oil.

A rapid, accurate, and efficient method for determining the sterol, uvaol, and erythrodiol concentrations was developed to meet International Olive Council (IOC) certification criteria for extra virgin olive oil (EVOO). The unsaponifiable fraction of the sample (0.2 g) was separated with a diatomaceous earth column, and the sterol and triterpenic dialcohols were isolated with a novel base-activated silica solid-phase extraction (SPE) cartridge cleanup protocol. The improved method and the IOC method provided identical pass/fail results (n = 34) for each of the six sterol and erythrodiol/uvaol IOC criteria used to assess olive oil. This method was validated, and recoveries of stigmasterol (88%) and β-sitosterol (84%) were greater than previously published values obtained using the IOC method. This method requires approximately one-third the time required to complete the IOC method and has great utility for the rapid screening of EVOO to detect adulteration, false labeling, and an inferior product.

Estrogenic Activity in Forages: Diagnostic Use of the Classical Mouse Uterine Bioassay

The classical mouse uterine bioassay was evaluated and adapted for routine diagnostic use in response to requests for evaluation of forages suspected of being estrogenic. Forages were extracted in acetone or 10% ethanol in acetone (v/v). Extracts were mixed with ground corn-based mouse feed. Immature female mice (n = 3/group) were fed a total of 100 g of the ground feed for 5 days. Body weights were monitored before and after the trial. After 6 days, the mice were euthanized and uterine weights were determined. Mean uterine weights were compared using 1 -way analysis of variance with preselected contrasts for individual means. Selected uteruses were fixed in 10% neutral buffered formalin for histologic examination. Control feeds, diethylstilbestrol (DES), estradiol, coumestrol, feeds with no reported estrogenic properties, and a feed that caused hyperestrogenism in cattle were tested. Moderate levels of estrogenic compounds resulted in dose-responsive uterine enlargements (10-270 ppm coumestrol over 5 days). Extremely high levels of estrogen frequently resulted in feed refusal and lack of uterine enlargement (10 ppm DES, 100 ppm estradiol). Diagnostically significant estrogenic activity was recovered from the feed known to have been estrogenic in cattle. The classical mouse uterine bioassay was relatively inexpensive, quick, repeatable, and capable of detecting clinically relevant coumestrol levels in hay.

Mineral, fiber, and total phenolic retention in eight fruits and vegetables: a comparison of refrigerated and frozen storage.

Minerals, total phenolics, and fiber were analyzed in several fruit and vegetable commodities to evaluate the differences between fresh and frozen produce. Magnesium, calcium, iron, zinc, and copper were evaluated in corn, carrots, broccoli, spinach, peas, green beans, strawberries, and blueberries. Each commodity was harvested fresh and split into two batches. Half of each commodity was kept fresh, and the other half was frozen. The nutrient content was analyzed over three storage times per treatment. The retention of nutrients was highly dependent on the commodity, but the majority of the commodities showed no significant difference between fresh and frozen for all analytes (p ≤ 0.05).

Gender and Single Nucleotide Polymorphisms in MTHFR, BHMT, SPTLC1, CRBP2, CETP, and SCARB1 Are Significant Predictors of Plasma Homocysteine Normalized by RBC Folate in Healthy Adults

Using linear regression models, we studied the main and 2-way interaction effects of the predictor variables gender, age, BMI, and 64 folate/vitamin B-12/homocysteine (Hcy)/lipid/cholesterol-related single nucleotide polymorphisms (SNP) on log-transformed plasma Hcy normalized by RBC folate measurements (nHcy) in 373 healthy Caucasian adults (50% women). Variable selection was conducted by stepwise Akaike information criterion or least angle regression and both methods led to the same final model. Significant predictors (where P values were adjusted for false discovery rate) included type of blood sample [whole blood (WB) vs. plasma-depleted WB; P < 0.001] used for folate analysis, gender (P < 0.001), and SNP in genes SPTLC1 (rs11790991; P = 0.040), CRBP2 (rs2118981; P < 0.001), BHMT (rs3733890; P = 0.019), and CETP (rs5882; P = 0.017). Significant 2-way interaction effects included gender × MTHFR (rs1801131; P = 0.012), gender × CRBP2 (rs2118981; P = 0.011), and gender × SCARB1 (rs83882; P = 0.003). The relation of nHcy concentrations with the significant SNP (SPTLC1, BHMT, CETP, CRBP2, MTHFR, and SCARB1) is of interest, especially because we surveyed the main and interaction effects in healthy adults, but it is an important area for future study. As discussed, understanding Hcy and genetic regulation is important, because Hcy may be related to inflammation, obesity, cardiovascular disease, and diabetes mellitus. We conclude that gender and SNP significantly affect nHcy.

Detoxification of Molinate Sulfoxide: Comparison of Spontaneous and Enzmatic Glutathione Conjugation Using Human and Rat Liver Cytosol

Previous lab studies implicated the sulfoxidation pathway of molinate metabolism to induce testicular toxicity. Once molinate is metabolized to molinate sulfoxide, it undergoes further phase II metabolism either spontaneously, enzyme catalyzed, or both to form glutathione-conjugated molinate. This study compared the metabolic capability of rat and human liver cytosol to form a glutathione (GSH)-conjugated metabolite of molinate. The GSH conjugation of molinate sulfoxide in rat cytosol was described by the constants Km of 305 μM and Vmax of 4.21 nmol/min/mg cytosol whereas the human values were 91 μM and 0.32 nmol/min/mg protein for Km and Vmax, respectively. At the same 1 mM GSH concentration, the in vitro bimolecular nonenzymatic rate constant of 3.02 × 10−6 μM −1 min−1 was calculated for GSH conjugation of molinate sulfoxide. Specific activity for rat and human glutathione transferase was calculated to equal 1.202 ± 0.25 and 0.809 ± 0.45 μmol/min/mg protein, respectively by 1-chloro-2,4-dinitrobenzene (CDNB) assay. Compared to a conventional GSH depletion model (BSO + DEM combination), molinate alone was nearly as effective in reducing GSH levels by approximately 90 and 25% in liver and testes, respectively. The impact of molinate sulfoxides ability to adduct glutathione transferase and inhibit the production of the glutathione conjugated metabolite was examined and found to be negligible.