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We compared the effectiveness of 2-hydroxy-4-(methylthio) butanoic acid (HMB) and DL-methionine (DLM) as sources of L-methionine activity in methionine-deficient primary cultures of pig liver cells and methionine-deficient early-weaned pigs. Viable hepatocytes were obtained from minced pig liver and maintained in a high density, differentiated, nonproliferation cell culture system. Culture medium was supplemented with HMB, DLM, or L-methionine, and cells were pulse-dosed with L-[14C(U)]leucine for 24 h to determine the level of protein synthesis. Leucine incorporation per milligram of protein indicated a six-to eightfold increase in protein synthesis (P < .01) with methionine levels between 5 and 10 microM, regardless of source of methionine activity. Two 24-pen replicate methionine dose titrations were conducted with 95 early-weaned commercial crossbred pigs. The pelleted corn, dried whey, and porcine plasma basal diet contained 1.5% lysine, .23% methionine, and .48% cystine and was supplemented with 0, .05, or .10% methionine activity as DLM or HMB for 21 d. There was a 134, 104, and 61% increase (P < .01) in cumulative ADG for each successive week on study with a 30 and 19% improvement in feed/gain (P < .01) after 7 and 14 d. Performance responses due to source of methionine activity did not differ and slope ratio potency determinations (gain vs intake of methionine source) of HMB vs DLM indicated a 119, 111, and 95% relative activity for cumulative weekly performance. These results support the hypothesis that HMB and DLM provide equimolar levels of methionine activity in swine.

The objective of the present experiments was to determine the biochemical basis for preliminary chick performance data, which indicate an ameliorative effect of 2-hydroxy-4-(methylthio)butanoic acid (HMB) when compared with DL-methionine (DLM) fed during hot conditions. In vitro passage of HMB or DLM across intact segments of small intestine from either control (thermoneutral, TN) or heat-stressed (HS) birds was used as a model for intestinal absorption. For DLM placed in the lumen, appearance in the outside buffer was reduced when using intestine from HS birds compared with tissue from TN birds. In contrast, the appearance of HMB in the outside buffer was greater using HS intestine, resulting in a substrate by environment interaction (P < .01). Slices of everted small intestine from TN and HS birds were used to study epithelial uptake of methyl labeled 14C-DLM by three transport pathways: diffusion, carrier-specific energy- and sodium-independent uptake (ESI), and carrier-specific energy- and sodium-dependent uptake (ESD). Correcting for extracellular volume, total epithelial uptake of 14C-DLM (diffusion plus ESI plus ESD) was reduced by 34% in HS intestine (P < .05). Energy-dependent uptake was observed to decrease by 87% in HS (P < .05). Energy-independent uptake was increased (136%, HS versus TN, P < .05), but not enough to compensate for the decrease in ESD uptake. Intestinal transport systems for glucose and leucine were also observed to change during HS, suggesting a role for cellular transport changes in the performance reduction associated with HS.

The objective of the present experiments was to determine whether the levels of supplemental methionine sources currently used in practical diets exceed the capacity of the chick to convert the supplement to L-methionine. Supplemental sources examined included DL-2-hydroxy-4-(methylthio(butanoic acid (DL-HMB, Alimet, or MHA) and DL-methionine (DL-Met). Two approaches were taken: first, the amount of enzyme activity available for conversion of the two supplemental methionine sources was determined using optimum reaction conditions for each and chick liver homogenate as the enzyme source. These experiments showed that total liver enzyme conversion activity was 564 mumol/h for DL-HMB and 529 mumol/h for DL-Met. The total activities for the two sources were not different when measured at saturating substrate concentration. Second, to address the question of whether the enzyme is limiting for either source under practical feeding conditions, birds were fed starter diets supplemented with DL-HMB or DL-Met at .25% of the diet for 3 wk. When hepatic levels of free HMB and methionine were determined, birds fed DL-HMB contained 7.6 nmol HMB/g of liver and 84.7 nmol methionine/g of liver. Birds fed DL-Met had levels of 7.6 nmol HMB/g liver and 80.3 nmol methionine/g liver. These results indicate no accumulation of HMB or DL-Met, which might occur if conversion capacity were saturated. By calculation, a bird consuming 100 g/day of a diet supplemented with .25% activity would need to convert about 70 mumol/h, indicating a sevenfold excess of enzyme in the liver alone.

Two subjects are discussed: first, the regulation of hepatic protein synthesis; and second, the intermediary metabolism of methionine, particularly with respect to the role of 2-hydroxy-4-(methylthio)-butanoic acid (HMB, Alimet). In the first section, the regulation of albumin synthesis is reviewed in terms of molecular events associated with the changes in albumin synthesis during fasting and refeeding. The effect of infection or of inflammatory stress on both albumin and total protein synthesis in the liver is also discussed. In the second part, research results are presented which indicate that HMB is a naturally occurring compound in methionine intermediary metabolism. The HMB synthesis by chick liver enzymes is demonstrated, and its role in normal avian methionine metabolism is discussed.

The objective of these experiments was to determine the origin of 2-hydroxy-4-(methylthio)-butanoic acid (HMB) detected in the liver and excreta of chicks that had never been fed Alimet (an 88% aqueous solution of HMB) or MHA (an 86% calcium salt of HMB). Gas chromatography and mass spectrometry were used to identify HMB in these birds. A normal biochemical pathway of 5'-deoxy-5'-methylthioadenosine (MTA) is proposed as the source of naturally occurring HMB. Studies of conversion of [methyl-14C]MTA to L-methionine by chick liver enzymesØe showed that radiolabeled HMB and 2-oxo-4-(methylthio)butanoic acid (keto-methionine) were synthesized during the reaction. Specific radioactivities of labeled HMB and keto-methionine showed that HMB is not synthesized from keto-methionine. Fractionation studies indicated that radiolabeled HMB formed from [14C]MTA could be used in the synthesis of L-methionine in the presence of peroxisomes and/or mitochondrial enzymes. In this way, HMB synthesized from MTA by chick liver enzymes functions as a precursor of L-methionine and as an intermediate in a naturally occurring pathway for the synthesis of L-methionine in the chick.

The purpose of these studies was to examine the role of the hindgut of the broiler chick in the absorption of 2-hydroxy-4-(methylthio)butanoic acid (HMB). When 14C-HMB was delivered directly into the hindgut, the rate of absorption from this gastrointestinal site was about 40% of the administered dose per hour. Plasma radiolabel appearance indicated that the 14C-HMB lost from the hindgut was being absorbed into the bloodstream of the bird. Decarboxylation experiments using cecal microorganisms showed that the loss of 14C-HMB could not be accounted for by bacterial metabolism. When birds were dosed with radiolabelled HMB and tissue samples were tested, results showed that the 14C-HMB that was absorbed from the hindgut was incorporated into protein in a dose-related manner. In addition, an equimolar, equal specific activity intraperitoneal dose of HMB did not alter the rate of HMB adsorption from the hindgut. This indicates that HMB absorption from the gut is not limited by HMB already in the body tissues. This result confirmed that the rate of HMB diffusion into the blood and its conversion to methionine in body tissues were sufficient to maintain the concentration gradient required for the continued absorption of HMB. Finally, whole body autoradiography comparing 35S-HMB and 35S-DL-methionine showed no substantial differences in terms of label density or distribution. These studies demonstrate that 14C-HMB disappears from the lumen of the large intestine and ceca when it is administered directly into the hindgut. This research confirms that HMB is absorbed throughout the entire gastrointestinal system.

Hydrolysis of 2-hydroxy-4-(methylthio)butanoic acid (HMB) dimer was observed in two model systems that were designed to emulate in vivo conditions. A physiologically feasible rate of hydrolysis of this methionine precursor has not previously been demonstrated in vivo. In the first model, simulated intestinal fluid (SIF) was used, and in the second model, sections of chick small intestine were incubated in vitro with the compound under study. When HMB dimer was incubated with SIF, hydrolysis was very rapid until half of the dimer was hydrolyzed. Subsequent hydrolysis was negligible in that model system. In contrast, hydrolysis neared completion when sections of small intestine were incubated with HMB dimer. These results indicate that studies of HMB dimer cleavage must include consideration of enzymatic hydrolysis factors.