Michael L. Bruss

Lipids and Ketones

This chapter covers the biochemistry and clinical chemistry of long-chain fatty acids, triacylglycerols, phospholipids, cholesterol, and ketones. The two most important functions of lipids are energy storage and membrane structure. Triacylglycerols are by far the most important lipids with regard to energy storage, and phospholipids and cholesterol are the most important lipid membrane constituents. Lipids serve other functions, including being precursors for steroids and bile acids (cholesterol), thermal insulation (triacylglycerols), and electrical insulation. Virtually all lipids are insoluble in water, which greatly complicates their handling in the body. Because of their insolubility, lipids must rely on proteins for transport for any significant distance in the body. This chapter begins with a discussion on long-chain fatty acids, and then discusses the structure, properties, and synthesis of triacylglycerol, and assays triacylglycerol. Structure and properties of phospholipids are also discussed. The chapter also elaborates properties of cholesterol and lipoproteins. The chapter concludes with a discussion on biochemistry of ketones including their synthesis and catabolism.

Effects of endotoxin infusion on circulating levels of eicosanoids, progesterone, cortisol, glucose and lactic acid, and abortion in pregnant cows

The effects of Escherichia coli endotoxin infusions (1.0 or 2.5 micrograms kg-1 over 6 h) on pregnancy were investigated in cows in the first, second and third trimester of gestation. Endotoxin increased the plasma levels of prostaglandins (PGs), thromboxane B2 and cortisol, and decreased progesterone. The severity of the clinical signs and the magnitude of the increases in plasma PGs, thromboxane B2 and cortisol tended to depend on the dose of endotoxin, but were independent of the gestation period. There was hyperglycemia followed by hypoglycemia and lactic acidemia. Hyperglycemia and lactic acidemia were significant only at the high dose of endotoxin. Endotoxin infusion at both doses caused a preferential mobilization of oleic acid from adipose tissue, and also had some effects on the mobilization of palmitic and stearic acids during the post-infusion period. The cows in the first trimester of gestation were more sensitive to the abortifacient effect of endotoxin than cows in the second and third trimester of gestation. The results of this study indicate that the mechanism of endotoxin-induced abortion in cows initially involves a prolonged release of PGF2 alpha and its subsequent stimulant effect on uterine smooth muscle contraction and luteolytic effect leading to a gradual decline in the plasma levels of progesterone. It was concluded that pregnancy terminates in the absence of an adequate level of progesterone, especially during the first trimester of gestation, when progesterone of extraluteal origin is not yet available, coupled with the PGF2 alpha-induced propulsive contraction of the uterus. In addition, the metabolic and circulatory failures in severe cases of endotoxemia, especially at the high dose of endotoxin, resulting either directly or indirectly via the release of various autacoids, catecholamines and cortisol, may also contribute to the termination of pregnancy at any stage of gestation.

Epidemiology and genetic basis of ketosis in Finnish Ayrshire cattle

The epidemiology and genetic variability of ketosis in Finnish Ayrshire cattle were examined. In the spring of 1982, a new health recording system was started on all dairy farms in Finland. Ketosis data collected during the first year were analyzed. Seven percent of 8201 Finnish Ayrshire cows which were daughters of 80 randomly selected bulls were treated for ketosis. Eighty-nine percent of cases occurred within 8 weeks after parturition, with the highest occurrence 3–5 weeks after calving. When cases were categorized by month of calving the risk of ketosis was higher during indoor feeding (September-May) than during outdoor feeding (June-August). Ketosis occurrence increased with parity, but was not associated with milk yield. Problems in estimating the correlation between ketosis and milk yield were discussed. The incidence rate among bull-daughter groups varied from 0 to 26 percent. The heritability estimate of 2.3% for ketosis on the binomial scale was not significant, even though it corresponded 81% on the normal scale. Research is needed on new statistical methods for threshold characters in order that breeding programs can be designed to accoint for possible inherited susceptibility to metabolic diseases.

Effects of intravenous administration of pirfenidone on horses with experimentally induced endotoxemia.

OBJECTIVE To characterize effects of IV administration of pirfenidone on clinical, biochemical, and hematologic variables and circulating tumor necrosis factor (TNF)-alpha concentrations in horses after infusion of a low dose of endotoxin. ANIMALS 18 healthy adult horses. PROCEDURES Horses were randomly assigned to 3 groups (n = 6 horses/group) and administered an IV infusion of 30 ng of endotoxin/kg or saline (0.9% NaCl) solution during a 30-minute period. Lipopolysaccharide-pirfenidone horses received endotoxin followed by pirfenidone (loading dose of 11.6 mg/kg and then constant rate infusion [CRI] at 9.9 mg/kg/h for 3 hours). Lipopolysaccharide-saline horses received endotoxin followed by infusion (loading dose and CRI for 3 hours) of saline solution. Saline-pirfenidone horses received saline solution followed by pirfenidone (loading dose and then CRI for 3 hours). Physical examination variables were recorded and blood samples collected at predetermined intervals throughout the 24-hour study period. Blood samples were used for CBCs, biochemical analyses, and determinations of TNF-alpha concentrations. RESULTS IV infusion of pirfenidone after administration of a low dose of endotoxin failed to attenuate the clinical, clinicopathologic, or cytokine alterations that developed secondary to endotoxin exposure. Intravenous infusion of pirfenidone after administration of saline solution induced mild transient clinical signs, but associated clinicopathologic changes were not detected. CONCLUSIONS AND CLINICAL RELEVANCE IV administration of pirfenidone was tolerated with only mild transient clinical adverse effects during infusion. However, administration of pirfenidone did not protect horses from the systemic effects of experimentally induced endotoxemia. Further studies of related, but more potent, drugs may be warranted.

Pharmacokinetics and metabolism of intravenous pirfenidone in sheep

Pirfenidone, a promising antifibrotic agent, was administered intravenously to six female sheep at 30 mg/kg. Four sheep received 14C‐pirfenidone simultaneously. Plasma and urine were obtained for assay of pirfenidone and its metabolites over two days, and tissues were obtained via necropsy. Samples were analysed for pirfenidone and metabolites using HPLC‐MS and flow scintillation spectrometry. Plasma pirfenidone disappeared with first order kinetics with a clearance of 1.2 l/kg/h, half‐life of 24 min, and distribution volume of 0.71 l/kg. After 48 h, the organs containing the largest quantity of 14C were lungs, liver and intestinal wall. Tissues with the highest concentration of 14C were lung, kidney, brain, liver, lymph node and adipose. Metabolites found in plasma and urine were hydroxypirfenidone (half‐life of 44 min) and carboxypirfenidone. Additional metabolites found in urine were hydroxypirfenidone glucuronide and acetoxypirfenidone. Approximately, 80% of the tracer eventually appeared in the urine, and approximately 50% of it was in the form of identifiable metabolites. Less than 1% of the dose appeared in the urine in the form of the parent drug. Quantitatively, most of the metabolites appeared in the urine within 2 h. Thus, the drug is rapidly and completely metabolized. Copyright

Chapter 4 – Lipids and Ketones

Publisher Summary This chapter covers the biochemistry and clinical chemistry of long-chain fatty acids, triacylglycerols, phospholipids, cholesterol, and ketones, a list including the majority of lipids found in vertebrates. Although lipids have many functions, two of the most important are energy storage and membrane structure. Triacylglycerols are by far the most important lipid with regard to energy storage, and phospholipids and cholesterol are the most important lipoid membrane constituents. Lipids serve other functions, including being precursors for steroids and bile acids (cholesterol), thermal insulation (triacylglycerols), and electrical insulation (various lipids). Virtually all lipids are insoluble in water, which greatly complicates their handling in the body. Because of their insolubility, lipids must rely on proteins for transport for any significant distance in the body, and various proteins have evolved to provide this function. The insolubility of lipids is an asset as well as a liability. Because of their insolubility, lipids generate no osmotic force, so large amounts of triacylglycerol can be stored in adipose without the weight gain due to water that would accompany it if it were soluble. The insolubility of lipids is vital to many of their functions in membranes.

Biliverdin reductase activity in cattle, sheep, rabbits and rats

1. Biliverdin reductase (BVR) activity was measured in post-microsomal supernatants of livers of cattle, sheep, rabbits and rats. BVR activities in bovine and ovine livers were 4.7 and 5.0%, respectively, of rat liver activity. 2. The finding of BVR activity in ruminants is in contrast to a previous report and may be due to the use of a different assay system. 3. Lapine liver had the lowest BVR activity of only 0.37% of rat liver activity. 4. Increasing the available heme by phenylhydrazine administration did not induce increased hepatic or splenic BVR activity in rabbits. 5. Maximal BVR activities were attained using NADPH as cofactor at pH 8.7 in sheep and rabbits and at pH 8.4 in cattle. 6. Differing concentrations of bovine or human albumins enhanced or inhibited BVR activity quite differently in the various species. 7. The finding of a very low, but measurable BVR activity in lapine liver and spleen may explain, in part, why rabbits, unlike rats, cattle and sheep, excrete primarily biliverdin (70%) into bile.

Effects of smooth and rough Pasteurella haemolytica lipopolysaccharides on plasma cyclic-nucleotides and free fatty acids in calves

The present study examined the potency of smooth or rough Pasteurella haemolytica lipopolysaccharide infusion (LPS, 24 ng kg-1 min-1 for 500 min) on plasma cyclic-nucleotides and several free fatty acids (FFA) in calves. Both smooth or rough LPS increased plasma cAMP immediately to its maximum at 1 h of infusion, whereas plasma cGMP levels rose slowly and peaked 12 h later. The increases in cAMP levels were more prolonged for smooth LPS than rough LPS. The maximum plasma cAMP rise coincided with increases of several plasma FFA. Rough LPS increased plasma oleic greater than palmitic greater than stearic greater than linoleic acids in the second hour and reached their steady state levels between 2 h of infusion and 5 h post-infusion. Thereafter, oleic acid remained maximally elevated, while stearic acid decreased and other FFA returned to baseline. Smooth LPS had no effects on palmitic and stearic acids, but elevated oleic acid in an essentially similar manner to rough LPS and increased linoleic acid initially at 5 h, followed by decreases throughout post-infusion. These results demonstrate that endotoxemia produces early marked elevations in plasma cAMP, a gradual rise in plasma cGMP and disproportionate increases in several plasma FFA. The data also demonstrate that smooth and rough LPS differ in their abilities to increase plasma cAMP and FFA and these may be attributed to differences in their in vivo mechanisms of action. The study suggests that cAMP and cGMP may mediate actions of endotoxin at the cellular level and that differences exist in release and/or utilization of each FFA at different stages of endotoxemia.

Hepatic bilirubin and UDP-glucuronate levels in bolivian squirrel monkeys exhibiting fasting hyperbilirubinemia

1. Bolivian squirrel monkeys (BoSMs), which are animal models for Gilberts syndrome, have 40% less hepatic bilirubin UDP-glucuronyltransferase (BR-UPPG-T) activity than Brazilian squirrel monkeys (BrSMs). 2. Although fasting results in similar decreases in hepatic UDP-glucose and UDP-glucuronate levels in both simian subspecies, increased activities (55%) of BR-UDPG-T are induced only in the fasted control BrSMs, which do not exhibit the marked fasting hyperbilirubinemia (FH). 3. Total hepatic bilirubin (BR) concentrations were 50% greater in both fed and fasted BoSMs when compared to BrSMs. 4. Hepatic unconjugated BR levels increase upon fasting only in Gilbert-like BoSMs, reaching concentrations twice that observed in BrSMs. 5. Elevated hepatic BR levels in fasted BoSMs may reflect BR overproduction or inadequate glucuronidation. 6. The increased BR-UDPG-T activity induced in BrSMs during fasting could compensate in-part for the UDPGA depletion and prevent the marked FH as observed in BoSMs.

Pharmacokinetics and clinical effects of pirfenidone administered intravenously in horses

OBJECTIVE To characterize the plasma pharmacokinetics and clinical effects of pirfenidone administered IV in healthy horses. ANIMALS 6 adult horses. PROCEDURES A 15 mg/kg dose of pirfenidone was administered IV over 5 minutes. Physical variables were recorded and blood samples collected prior to infusion; 2.5 minutes after beginning infusion; at the end of infusion; and at 3, 6, 9, 12, 15, 20, 25, 30, 40, 50, 60, 75, and 90 minutes and 2, 2.5, 3, 4, 6, 8, 12, and 24 hours after completion of infusion. Plasma concentrations of pirfenidone and its metabolites were determined. RESULTS Mild clinical effects, including tachycardia and muscle fasciculations, were observed during drug administration but stopped at the end of the infusion. Pirfenidone and 2 metabolites, hydroxypirfenidone and carboxypirfenidone, were detected by the end of the 5-minute infusion. Mean peak plasma concentration of pirfenidone was 182.5 micromol/L, detected at the end of the infusion. Mean peak plasma concentrations of hydroxypirfenidone and carboxypirfenidone were 1.07 and 3.4 micromol/L, respectively, at 40 minutes after infusion. No parent drug or metabolites were detected at 24 hours. Distribution of pirfenidone best fit a 2-compartment model, and the drug had mean +/- SEM elimination half-life of 86.0 +/- 4.7 minutes, mean body clearance of 6.54 +/- 0.45 mL/kg/min, and apparent volume of distribution at steady state of 0.791 +/- 0.056 L/kg. CONCLUSIONS AND CLINICAL RELEVANCE Intravenous administration of pirfenidone was tolerated with transient adverse affects during infusion, and drug clearance was rapid.