)V1ESI_} RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from “ your record. FINES wiH be charged if‘book is returned after the date stamped be10w. NT METHYL HISTIDINE CONTENT OF FEEDSTUFFS FED TO RUMINANTS By Phoebe Wei-Tsu Gur-Chiang A THESIS Submitted to Michigan State University in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE Department of Animal Science 198U C>1985 PHOEBE WEI-TSU GUR-CHIANG All Rights Reserved ABSTRACT NT METHYL HISTIDINE CONTENT OF FEEDSTUFFS FED TO RUMINANTS By Phoebe Wei-Tsu Gur—Chiang The NT—methylhistidine content of 12 feedstuffs, duodenal digesta from steers fed alfalfa haylage and u different muscles obtained from young and finished steers was determined. Degradation of NT-methylhistidine by ruminal microorganisms was studied in zitrg. NT-Methylhistidine was quantitated using ion-exchange chromatography. Results indicate that feeds consumed by ruminants contain little or no NT-methylhistidine. Steers, consuming dry matter at 2-3% of bodyweight would consume 5—15 umol NT—methylhistidine daily. in 31339 degradation of NT-methylhistidine by rumen microbes was 1U.5% while leucine (control) was totally degraded. Skeletal muscle NT-methylhistic ne content was higher in the older steers (3.5 umol /g protein ; 2.8 umol /g protein) while red' muscles contained less NT-methylhistidine than predominantly white muscles (2.9 umol /g protein ; 3.U umol /g protein). ACKNOWLEDGMENTS The author would like to express her deepest appreciation and thanks those people who have supported me and were helpful throughout the duration of this NS graduate program. Dr. Werner G. Bergen, serving as my major professor, always encouraged and guided me and my whole graduate committee, Drs. Bergen, Robert A. Merkel, Milo B. Tesar, provided me with the things that were needed in my master's research, and challenged me to complete the research work outlined and to obtain in depth knowlege in animal nutrition. Sincere thanks is extended to Dr. R. H. Nelson, Past Chairman of the Department of Animal Science and Dr. M. G. Hogberg, Chairman of the Department of Animal Science for providing necessary research facilities and supplying excellent research circumstances. In addition, I am deeply grateful to Mrs. Elizabath Bimpau for her unceasing assistance with my research, correction of this manuscript and her friendship. Sincere thanks is also extended to Dr. Pao Kwen Ku for his comments and statistical advice in preparation of this thesis. I also would like to express a very special thanks to my fellow graduate students, Patty Dickerson and Kristen Johnson, for they always gladly assisted me with collection of samples and their precious friendship. I wish to thank my beloved parents, Mr. and Mrs. Hsio-Tien Chiang and parents-in-law Mr. and Mrs. Yin-Yu Gur and my family in my country, The Republic of China, for their love and confidence in me. Their continued support and diligent praying has been a source of motivation in completing this work. Above all, I wish to express my deepest and heartfelt thanks to my loving husband Yain-Tain Gur, I will never forget his love and support. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF APPENDICES INTRODUCTION REVIEW OF LITERATURE I. NT—Methylhistidine Historical Background A. Nomenclature and Structure B. Initial Work . . . . . . C. Further Development a. b. C. Q Site Of iHistidine Methylation N -Methylhistidine as an Index of Muscle Protein Breakdown . Location of N -Methylhistidine in Muscle Myofibrillar Proteins . . Metabolism of N -Methylhistidine Distribution of N -Methylhistidine in Tissue from Different Animals . NT—Methylhistidine in Plants or Microorganism . . Analysis of NT—Methylhistidine —— Early and Newer Methods . . . . . . . MATERIALS AND METHODS I. Pyridine-Column Chromatography Procedure II. NT—Methylhistidine Analysis by Ion Exchange Chromatography and Total Analytical System Recovery III. Analysis Technique for Feedstuffs and NT— Methylhistidine Recovery Procedure from Steers IV. NT—Methylhistidine Analysis of Duodenal Digesta H- JI'EJZ-‘UU LA) UL) l3 17 22 26 27 32 33 33 35 38 V. Ruminal Degradation of NT-Methylhistidine In Vitro . . . . . . . . . . . . . VI. NT-Methylhistidine Content of Bovine Muscles VII. Statistical Analysis . RESULTS AND DISCUSSION . . . . . . . . . . . I. Pyridine Column Chromatography . . . II. Amino Acid Analysis and NT—Methylhistidine Recovery . . . . . . . . . . . . . . . . . III. The Concentration of NT— Methylhistidine in Feeds uffs . . . A. N -Methylhistidine Recovery in Alfalfa Hay Samples . . . . . . . . B. NT-Methylhistidine in Feedstuffs . IV. N -Methylhistidine Content in Duodenal Digesta of Steers . . . . . . . . . . . . . . . . V. N -Methylhistidine Degradation by Ruminal Microorganism In Vitro . . . . . VI. NT-Methylhistidine in Bovine Muscles . . CONCLUSION . . . . . . . . . . . . . . . . . . . . APPENDICES . . . . LIST OF REFERENCES . . . . . . . . . . }.Jo Fl “3 62 62 62 69 73 78 87 88 93 Table Table Table Table Table Table Table Table Table Table Table Table 10. ll. 12. LIST OF TABLES Characteristics of Amino Acid for In Vivo Measurements of Muscle Protein Breakdown Methylated Amino Acids in Muscle . . NT—Methylhistidine Content of Actin and Myosin Prepared from Different Muscles NT-Methylhistidine Content of Actin and Myosin from Different Species . . . . 8201 Supplement Composition NT—Methylhistidine Recovery from Dowex BOW-X8 Column . . . . . . . . . . NT-Methylhistidine Recovery of Alfalfa Hay Crude Protein and NT—Methylhistidine of Feedstuffs . . . . . . . . . . . . . Calculation of Urinary NT-Methylhistidine Excretion in a Steer Fed Dried Orchard-Bromegrass Hay N -Methy1histidine, Nitrogen and Crude Protein Content in Duodenal Digesta from Steers . . . . . . . . . . . The Degradation of BCAA- Leucine and N -Methylhistidine After Incubation for 8 Hours of Rumen Fluid Collected Before Feeding and A Hours After Feeding The NT-Methylhistidine Content in Young and Finished Steers . . . . . . . iii Page 10 21 23 25 A0 55 63 6A 66 77 79 Table 13. Table 1“. Table 15. The NT-Methylhistidine Content at Different Ages of Animals . . . NT-Methylhistidine Content of Mixed Proteins in Muscles from Human . NT-Methylhistidine Content of Myosin iv Figure Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES Flow of Amino Acids in the Intact Organism, Illustrating Inter- and Intracellular Amino Acid Reutilization Amino Acid Sequences Around the Single N —Methy1histidine in Actin and Myosin Schematic Depiction of the Location of N -Methy1histidine in Myosin and Actin The Origin and the Fate of N —Methylhistidine in Myofibrillar Proteins . . . . . . . . Histidine Dipeptides and Metabolism of N -Methylhistidine . . . . . Dowex BOW-X8 Chromatography of N —Methylhistidine, Histidine andchPA with Pyridine— Water Eluents . . . . The Chromatogram of 100 ml .21“? Pyridine, Elution in Amino Acid Analyzer The Chromatogram of Initial 125 ml 1 M 1A 16 18 19 NA “7 Pyridine Elution with N -Methy1histidine Elutes in “0- 60 ml Fraction . . . The Chromatogram with Histidine Elutes in 80— 125 ml Fraction of 125 ml 1 M Pyridine Figure 9. Elution Figure 10. The Chromatogram of an Additional 100 ml 1 M Pyridine Figure 11. Chromatogram of Combination of .1 mM N -Methylhistidine, .1 mMa GPA and 10 mM Histidine Mixed 1/1/1 (V/V/V) , Figure Figure Figure Figure Figure Figure l2. 13. I“. 15. 16. 17. Page Chromatogram of .1 mM aGPA and 10 mM Histidine . . . . . . . . . . . . . . . 58 Chromatogram of .1 mM NT-Methylhistidine and .1 mM aGPA . . . . . . . . . . . . . 60 Chromatogram of 25 mg Protein Timothy Hay . . . . . . . . . . . . . . . . . . 67 Chromatogram of External "Spike" of NT-Methylhistidine 25 mg Protein Timothy Hay o o o o o o o o o o o o o 70 Chromatogram of Duodenal Digesta Sample 7“ Chromatogram of Bovine Muscle Sample . . 8“ vi LIST OF APPENDICES Page Table A. l Ninhydrin Solution Preparation . . . 93 Table A. 2. Charcoal Column Preparation Procedure 9U Table A. 3 Composition of Ohio Media . . . . . 95 Table A. M Composition of Mineral Mix "0" . . . 96 Table A. 5 Intake, Flow of Dry Matter . . . . . 97 vii INTRODUCTION There has been a growing interest in recent years in the study of protein turnover in ruminants (McCarthy, 1981). To assess the role of dietary and endocrine status on protein turnover (breakdown rate; BR) in large domestic animals it is convenient to employ noninvasive procedures utilizing a muscle protein turnover marker; one amino acid NT-methylhistidine is such a marker. Fifteen years ago, NT-methylhistidine (3-methylhistidine; 3-MeHis) was identified as a constituent of both actin (Asatoor & Armstrong, 1967) and myosin (Johnson et al., 1967). The available evidence indicates that at a stage during the synthesis of the polypeptide chains of actin and myosin, some of the peptide-bond histidine residues are methylated to give NT—methylhistidine (Haverberg, 1975; Ward and Buttery, 1978). NT—Methylhistidine cannot be reutilized for protein biosynthesis when released by protein breakdown and is excreted quantitatively in the urine. Asatoor and Armstrong (1966) were first to suggest that the measurement of the rate of excretion of NT-methylhistidine could provide an estimate of myofibrillar degradation or turnover. Since 2 then, many researchers have applied this concept and utilized NT—methylhistidine to assess skeletal muscle ER in man and animals (Young and Munro, 1978). To utilize NT—methylhistidine as a skeletal muscle protein turnover marker, it is essential that all nonendogenous (i.e. exogenous) sources of NT-methylhistidine be eliminiated. It had been suggested that forages and other feeds fed to ruminants may contain sizable levels of NT-methylhistidine. This research was initiated to determine the concentration of NT-methylhistidine in 12 different feedstuffs, to assess the ruminal microbial metabolism of free NT-methylhistidine and to assess duodenal flow of NT-methylhistidine in cattle. To obtain these data, extensive work had to be done on analytical techniques for NT-methylhistidine. REVIEW OF LITERATURE I. NT-Methylhistidine Historical Background A. Nomenclature and Structure Alpha amino- B—(l-methyl-H-imidazole) propionic acid was assigned the name tele—methylhistidine (NT-methyihistidine) by the IUPAC-IUB (1972) to remove confusion as to which nitrogen on imidazole—ring the methyl group of NT-methylhistdine is attached. NT-Methylhistidine is an isomer of Nfl-methylhistidine (l-methylhistidine; a-amino-B-(1-methyl-5—imidazole) propionic acid). The chemical structure of NT-methylhistidine and N"-methylhistidine are as follows: HC =C—- CH2-—- CH—COOH I l \C/ NT—Methylhistidine (3—methy1histidine) H3C—- NH2 HC===:C-——-CH2-——-CH-——-COOH N N-——-CH \CH/ 3 NW—Methylhistidine (1—methylhistidine) NH2 B. Initial Work Urinary excretion of N methyl derivatives of histidine had been observed by early workers. Searle and Westall (1951) showed that 1-methy1histidine is a normal urinary constituent. In normal man, amino acids containing the imidazole ring account for 30% of the free amino acid a-amino nitrogen found in the urine. NT—Methylhistidine is usually found in smaller amounts than the 1-methylhistidine (Tallan et al., l95U). l—Methylhistidine has long been known to be a constituent of anserine and had been isolated in the free form from urine (Searle and Westall, 1951). During this time Tallan et a1. (19SU) were also working on the isolation of NT-methylhistidine from cat urine. Both laboratories demonstrated that NT—methylhistidine from man or cat were indistinguishable when (co)chromatographed on paper and on Dowex 50. NT—Methylhistidine has been synthesized from phthaloylhistidine and from L—histidine. Synthetic NT-methylhistidine, identical with the natural product in respect to elementary analysis, optical rotation, chromatographic behavior, and infra-red spectrum, was isolated from the reaction mixture by chromatography (Tallan et al., 195“). C. Further Developments a. Site of Histidine Methylation Datta and Harris (1951) showed that after the ingestion 5 of large amounts of rabbit meat by humans (rabbit muscle contains high amounts of anserine) there was a measurable rise in the excretion of 1—methylhistidine but not for NT-methylhistidine. Tallan et a1. (195M) could not locate the source of the urinary NT-methylhistidine both in the cat and in the human. Asatoor and Armstrong (1967) demonstrated that in samples of blood and urine collected under fasting conditions NT—methylhistidine was present and therefore urinary NT-methylhistidine was thought to be of endogenous origin. Asatoor and Armstrong (1967) presented evidence that NT-methylhistidine is a component of the muscle protein actin (now we know that actin is found in all cells). All acid hydrolysates of actin preparations (actin was prepared by the procedure of starch gel electrophoresis (Carsten and Mommaerts, 1963) ) contained NT—methylhistidine in the proportion of 1 mole per 8 to 10 moles of histidine. Asatoor and Armstrong (1967) also showed that histidine and the methyl group of methionine serve as precursors for NT-methylhistidine, and that urinary l—methylhistidine and NT-methylhistidine come from pools with markedly different turnover rates. Johnson et a1. (1967) provided evidence for the presence of NT—methylhistidine as part of the primary structure of actin and myosin. Their work also confirmed the Asatoor and Armstrong (1967) findings on the presence of NT-methylhistidine in actin hydrolysates. NT—Methylhistidine occurs as a constituent of skeletal 6 and cardiac muscle actin and in the myosin of skeletal muscle consisting predominantly of white fiber types (Lobley and Harris, 1977). NT—Methylhistidine is present in actin from a number of species of adult and fetal skeletal muscle, whereas it is only found in myosin shortly after birth in the rabbit (Johnson et al., 1969). Trayer et al. (1968) also pointed out that the ninhydrin-positive material corresponding to NT-methylhistidine could not be detected in significant amounts in acid hydrolysates of myosin isolated from skeletal muscle of the 28 day old fetus. Actin from red skeletal muscle and cardiac muscles of the rabbit, from smooth muscle of the cow uterus and from certain invertebrate muscle gave a NT-methylhistidine: histidine ratio as found in purified actin from adult rabbit white skeletal muscle. Myosins from red and cardiac muscle had a much lower NT-methylhistidine: histidine ratio than that of purified myosin from adult rabbit white skeletal muscle (Johnson et al., 1969). Young et al. (1970) showed that NT—methylhistidine was not bound to transfer RNA by amino acyl ligase from skeletal muscle in vitrg_and thus the methylation process must occur at a stage in fibrillar protein synthesis which is subsequent to the formation of histidyl-tRNA. Since methylation occurs after translation, reutilization is not possible, and that free NT—methylhistidine cannot be directly used for the synthesis of the fibrillar proteins. And during the course of actin and myosin breakdown it is 7 liberated into the free amino acid pool and then quantitatively eliminated from the body, chiefly via the urine (Young et al., 1972). These findings were consistent with the observations of Asatoor and Armstrong (1967) that the specific activity of uniformly 1“ C-labeled histidine is unchanged after isolation of injected label as NT-methylhistidine from muscle homogenates. Young and Munro (1978) indicated that studies of tissue protein breakdown in 1219 are difficult because of extensive amino acid reutilization as shown in Figure l. The amino acids that are released during the intracellular breakdown of proteins can be extensively reutilized for protein synthesis within the cell (intracellular recycling) or they may be transported to other organs where they enter pathways of protein anabolism (intercellular recycling). This recycling frustrates studies of protein breakdown after administration of a labeled amino acid followed by measurement of the rate of loss of label from the tissue proteins. b. NT—Methylhistidine as an Index of Muscle Protein Breakdown Although no rapid and satisfactory method has yet been _devised for measurement of protein degradation in the whole animal the work of Young and his colleagues (1973) indicates that in vivo catabolism of muscle can be followed in the urinary elimination of NT-methylhistidine (Lobley & Figure 1. Flow of Amino Acids in the Intact Organism, Illustrating Inter- and Intracellular Amino Acid Reutilizationa [Tissue A Synthisis r4Proteii Breakdown ,Jk Intracellular AA:5 'AA Amino Acid Uptake of ’///V 7 Reutilization Secreted ' _(Protein) Proteins —"' 1 r-A—‘I -—r- «hf— Blood l I) l Amino LK/Acids Intercellular Plasma ‘ ‘ Amino Acid Proteins : ‘} ‘ Reutilization [L M .1 I Tissue B i ‘ AA‘tI AIIAA Intracellular 1.1 Amino Acid Protein Reutilization Protein J Secretion a Adapted from Young and Munro (1978). 9 Harris, 1977). Young and Munro (1978) recognized the potential importance of previous findings on myofibrillar NT-methylhistidine content and also suggested that the urinary excretion rate of NT-methylhistidine could potentially be used as an index of muscle protein breakdown provided that: (A) The NT-methylhistidine was present exclusively in muscle protein and at a constant amount. (B) NT-Methylhistidine released after protein degradation was neither reutilized for protein synthesis nor metabolized. (C)I Free NT-methylhistidine was rapidly and quantitatively excreted in the urine. Initial experiments apparently confirmed all three conditions. Young and Munro (1978) also listed characteristics of an ideal marker amino acid as shown in Table 1. Asatoor and Armstrong (197M) proposed and Young and Munro (1978) Justified and clarified how NT—methylhistidine in muscle fulfills these characteristics (Table 1). Young et a1. (1973) first discussed the potential use of NT-methylhistidine excretion as an index of progressive reduction in muscle protein catabolism during starvation in obese human patients. Similar studies were done by Rao and Nagabhushan (1973) in men suffering from protein- calorie malnutrition. These two groups observed a progressive decrease in urinary NT—methylhistidine with Table l. a 10 Characteristics of Amino Acid for In_Vivo Measurements of Muscle Protein Breakdowna Amino Acid is modified chemically after peptide bond synthesis. Chemically modified group does not undergo exchange once it appears in the protein-bound amino acid. Concentration is known or constant in muscle protein. Amino acid not formed to an important extent in other tissues. Released at the same time as other amino acids are released from the completed protein. Not reutilized for protein synthesis. Does not undergo further metabolism in the bodv. Has a low renal threshold. ~ Quantitatively excreted in the urine. Adapted from Young and Munro (1978). ll starvation and protein-calorie malnutrition. These findings suggested that the reduced output of urinarv NT—methylhistidine coincided with decreased urinary N output due to an adaptation and decrease in catabolism of muscle proteins as starvation and protein-calorie malnutrition progressed (Millward et al., 1976). Haverberg et al. (1975) using rats, showed a marked and progressive decrease in NT-methylhistidine excretion in the protein-depleted group. The group restricted in both dietary protein and energy, showed an initial small increase in daily NT-methylhistidine output but then a decrease in excretion. These results were confirmed by Funabiki et al. (1976) and Nishizawa et a1. (1978), in subsequent studies. Ogata et a1. (1978) and Nishizawa et a1. (1978) both showed an increase in NT-methvlhistidine excretion initially in starved rats, indicating an increase in degradation of muscle. Ogata et a1. (1978) did not observe a decrease.in NT-methylhistidine excretion, however in their study rats were starved for 72 hours then fed for 2H hours. Nishizawa et al. (1978) did observe a decrease in excretion of NT—methylhistidine after A days of starvation. Omstedt et a1. (1978) used NT-methylhistidine excretion as an index for muscle degradation in evaluating protein quality and found that the better the protein quality the greater was the rate of growth of the rat and the higher was the total urinary NT-methylhistidine (nonacetylated + acetylated) excretion. 12 NT- Methylhistidine, unfortunately cannot be used as a protein turnover marker in all species. Harris and Milne (1980, 1981b) demonstrated that urinary NT—methylhistidine excretion is an inadequate index of muscle protein breakdown in the pig and sheep. However, by the criterion of the rate of clearance of labeled NT-methylhistidine from the body, the excretion of NT-methylhistidine in urine appears to be a Valid index of muscle protein breakdown in cattle (Harris and Milne, 1981a; McCarthy et al., 1983) as well as man and laboratory rodents (Young and Munro, 1978). McCarthy et al. (1983) found that NT-methylhistidine was quantitatively excreted by cattle at a rapid rate, thus demonstrating the NT-methylhistidine released during breakdown of muscle proteins would be quickly removed from the body. However, two problems in the use of NT-methylhistidine as a muscle protein turnover marker were not directly addressed by McCarthy et a1. (1983). The first problem is the possibility that NT-methylhistidine of exogenous feed origin may be absorbed and excreted in the urine and a second problem is the NT—methylhistidine contribution from other contractile proteins of non—muscle tissue whose turnover may differ from skeletal muscle proteins (Rennie et al., 1983)." McCarthy et al. (1983) assuming that only 80% of urinary NT-methylhistidine excretion arose from skeletal muscle tissue, demonstrated that NT—methylhistidine excretion per unit of body weight 13 did not differ between finishing steers of large and small frame sizes and that the fractional degradation of muscle protein did not differ between large and small cattle. The fractional rate of muscle degradation was also shown to decline with age for all steers. 0. Location of NT- Methylhistidine in Muscle Myofibrillar Proteins Huszar and Elzinga (1971) determined the amino acid sequence around the single NT-methylhistidine residue in rabbit skeletal muscle myosin by the use of the enzyme thermolysin. This enzyme usually hydrolyzed peptide bonds in which the amide bond is contributed by an amino acid with a hydrophobic side chain, such as Gly-Ile and Thr-Tyr bonds. Thermolysin does not split the Asp-Val bond. Elzinga (1971) determined the amino acid sequences around NT-methylhistidine in both myosin and actin; his results are presented in Figure 2. In summarizing these studies, Huszar et a1. (1971) indicated that: l) Actin and myosin are the only myofibrillar proteins that have been shown to contain NT-methylhistidine; this methylated amino acid is located in the globular subfragment—l of myosin. The only other proteins which have been reported (at that time) to contain this residue was ameba actin (Weihing et al., 1969). Figure 2. 19 Amino Acid Sequences Around the Single NT -Methylhistidine in Actin and Myosina Myosin : Leu-Leu-Gly-Ser—Ile-Asp—Val-Asp— NT -Methy1hist idine-Gln—Thr—Tyr—Lys Actin : Leu-Thr—Leu-Lys-Tyr-Pro-I1e-Glu- NI -Methylhistidine-'Irp -Gly-Ile-I1e aAdapted from Elzinga (1971), Huszar and Elzinga (1981). 15 2) Actin and myosin subfragment-l contain sites of interaction with nucleotides and metals. Huszar et al. (1971) further pointed out that a feature common to both peptides is the acidic residue adjacent to NT—methylhistidine; acidic side chains often lie at the surface of a protein, so the side chains of NT—methylhistidine would be at or near the surface of actin and myosin. Elzinga et al. (1973) published the complete amino acid sequences of actin of rabbit skeletal muscle. The actin polypeptide chain is composed of 37A residues, including one residue of NT—methylhistidine. Their study represented the first complete determination of the amino acid sequence of a myofibrillar protein. The schematic depiction of the location of NT-methylhistidine in myosin and actin is shown in Figure 3 (Young and Munro, 1978). Haverberg et al. (197“) demonstrated that there were no significant age-related changes in the NT—methylhistidine content of either actin or myosin in rats weighing from 9A to 32“ g, but Young et a1. (1972) reported that in rats the relative proportion of NT-methylhistidine and its acetylated metabolite, NT—acetyl methylhistidine, in urine changed with increasing body weight in growing rats. The N-acetyl form accounts for the major fraction of total urinary NT—methylhistidine in the adult rat (Young et al., 1972) and at present, this acetyl form is only found in laboratory rodents (rats and mice). N-Acetylation of NT-methylhistidine has not been observed in other animals 16 Figure 3. Schematic Depiction of the Location of NT-Methylhistidine in Myosin and Actina Myosin NT-Methylhistidine (1 mole per heavy chain) E-N-Monomethyllysine (1 mole per heavy chain) E-N-Trimethyllvsine (2 mole per heavy chain) *— LMM 9 HMM' ‘3”;9 s-2 " Actin a Adapted from Young and Munro (1978). bRabbit 37“ residues. 17 (Young et al., 1978; McCarthy, 1981). Haverberg et a1. (1975) studied the concentration of NT-methylhistidine in blood serum and in the mixed proteins of heart, brain, lung, kidney, diaphragm, spleen, testis, stomach, liver and leg skeletal muscles of approximately “00 g body weight male rats. Haverberg et a1. (1975) indicated that skeletal muscle is likely to be the major source (658 nmol NT-methylhistidine/g tissue) of urinary NT-methylhistidine, which in turn is then a reflection of myofibrillar protein breakdown in skeletal muscle. d. Metabolism of NT—methylhistidine Young and Munro (1978) presented a summary of NT-methylhistidine metabolism in animals (Figure A). Figure A shows the relationship between the NT—methylhistidine present in actin and myosin of skeletal muscle and urinary output of the amino acid in rats and human subjects. The NT-methylhistidine released during protein turnover is excreted quantitatively in urine. While ultimalteyl all NT-methylhistidine synthesized and released from muscle protein is quantitatively excreted in the urine and not catabolized within the body, the imidazole amino acids tend to form specific dipeptides that are found in the blood and tissue of the animals (Figure 5, Carnegie et al., 198“). The dipeptides present an intermediate step in NT-methylhistidine metabolism and cause spurious and slow urinary excretion of the amino acid 18 Figure A. The Origin and the Fate of NT-Methylhistidine in Myofibrillar Proteinsa SAME Muscle ‘*“‘ Methionine 1r}; 1. lrji/ Protein (Actin Synthesis jand Myosin) 2_ Amino jfBreakdown Protein .r "' " (Recycling) Acids + N Niggiéine Breakdown Transamination and 3. Oxidation l f Release 9 | Blood Amino Acids V N’T-Methylhistidine Liver , NeAcetyl- 5 539931- N -Methyl- u ' _1_ histidine ' - t 4, M 4— Excertlon NT-Methvlhistidine Hodificaoion (Rat) Urine NT-Methylhistidine (Major form in man) N —Acetyl—NT-Methylhistidine (Rat) a Adapted from Young and Munro (1978). Figure 5. 19 Histidine Dipeptides and Metabolism of Nr-Methylhistidinea Muscle ----- -> NI —Methylhistidine -----> Urine Not Metabolized By pass pool Dipeptide Histidine Dipeptides Anserine B-alanly-l-methylhistidine Balenine B-alanly-L-Nr~methylhistidine Carnosine B-alanyl—histidine a Adapted from Carnegie et al., (198“). 20 such that the rate of urinary NT—methylhistidine excretion cannot be used to assess muscle protein turnover. In sheep, Harris and Milne (1981a, b) reported evidence of a NT-methylhistidine dipeptide, called balenine (Figure 5), present in muscle which ties up large amounts of NT-methylhistidine released during muscle protein degradation into a storage—bypass pool which presumably has its own kinetic propeties. Balenine (B-alanyl—L—NT-methylhistidine) is present in skin and muscles of whales, snakes, pigs with lower amounts in chicken, cattle or sheep (Carnegie et al., 198D). Anserine (B-alanyl-L-l-methylhistidine) occurs in much higher levels in muscle of chicken, steers and Cattle than pigs. The function of the histidine dipeptides is not known (Young and Munro, 1978). Bergen and Potter (1971) pointed out that while the basic amino acids l-methylhistidine, NT-methylhistidine and the dipeptide carnosine (B-alanyl-L—histidine) are present inplasma of sheep, another major methylated amino acids eluted with lysine. This amino acid was identified as N methyl lysine (NML) and is present in amounts exceeding the methylated histidine. NML is not found to a great extent in cattle and cannot be used as a protein turnOVer marker as NML is the carnitine precursor in animals (Horne et al., 1971; Tanphaichitr et al., 1971; Horne and Broquist, 1972; Borum and Broquist, 1977). Table 2 lists 21 Table 2. Methylated Amino Acids in Musclea NT-Methylhistidine (3—Methy1histidine) Soluble dipeptide: Balenine (B-alanyl-l-NT- methylhistidine) in skin and muscles of whales, snakes and pigs. Protein-bound: Myosin (white or fast twitch fibers) Actin (all muscles and cytoplasmic actins) 1-Methylhistidine (Nfl-Methylhistidine) Soluble dipeptide: Anserine (B-alanyl-l- methylhistidine) Function: ATPase activators Histamine-like action Buffer action e-N-Monomethyl lysine and e -N—trimethyllysine N5 —N5 -Dimethylarginine a Adapted from Young and Munro, 1978. 22 the methylated amino acids that have been isolated in muscle (Young and Munro, 1978). e. Distribution of NT-Methylhistidine in Tissue from Different Animals Johnson and Perry (1970) were concerned with the distribution of NT—methylhistidine in myosin and actin from various species and muscle types. Mixed longissimus dorsi and leg muscle of adult New Zealand White and Dutch rabbits, pigeon breast muscle, adult rabbit cardiac muscle, mixed skeletal muscles of white mice, smooth muscle of cow uterus, crab claw muscle and lobster tail muscles were analyzed for NT-methylhistidine content (Johnson and Perry, 1970). In rabbits, the amounts of NT-methylhistidine in cardiac and red skeletal muscle were significantly lower than that present in adult mixed skeletal muscle, which is mainly white. NT-Methylhistidine contents of the mixed skeletal muscles from mice was also much lower than that of mixed skeletal muscle of the rabbit (Table 3; Johnson and Perry, 1970). The fact that NT-methylhistidine is present in actin and myosin isolated from seven species of vertebrates and invertebrates strongly suggests that it is a normal component of these proteins in all species. The NT—methylhistidine content of actin, however, appears to be much more constant than is the case with myosin. The values for the NT-methylhistidine content of myosin were much more variable than those of actin and in all cases the 23 .onma .mgpom new cowCQOh Eopm popomp¢m s2. mm.m m.mHHH m.mm”H I om.m I ~.mmufi ammo popmooq pmpmfisom I mm.m I m.:m"H zmao pogo pumpomppo>cH I mo.a I m.ww"H mm. m.m :.wua :.omua mason: Zoo zoooEm I mu. I mafia Hmpmaoxm pmxHE omsoz me. an. QHHH mmaufi ammo: pfionmm I Ho. I munfi ammopn commam pmpmflhwm pom Hmuoamxm pwxfie mo. mm.H m.nna m.z:”H panama uH3p< poomfigum mpfizz capo< cflmosz cauo< :Hmomz oopsom ooze Acfiopopo ocfipfipmam Lo Hoe Loo wospfimomv pcopcoo oaomsz ocfipfipmficfizcuozIez oCfipprHQH>cpozIez wofiomzz economcfia EoLp cosmoopm camozz pom sfipo¢ mo pcoucoo mcpfifipmfizfimzquIez .m magma .m 2n NT-methylhistidine/histidine ratio of actin is higher. The NT-methylhistidine/histidine ratio of myosin varies from 1:20.U (Cow uterus) to 1:178 (Rabbit heart), however NT—methylhistidine/histidine ratio of actin varies from 1:7.6 (Adult rabbit mixed skeletal) to 1:15.2 (Lobster tail) (Johnson and Perry, 1970; Table 3). Johnson and Perry (1970) concluded that 1) the NT-methylhistidine content is highest in myosin from white skeletal muscle and lower values are obtained from myosin of red skeletal and smooth muscle, and 2) the NT—methylhistidine content of actin was similar in all of the types of muscle from which it was isolated. Haverberg et al. (197”) determined the content of NT-methylhistidine in act n and myosin from different species (Table A). Coher tissues contain actin and therefore have NT-methylhistidine, at least 16.6% of the daily urinary NT-methylhistidine excretion is released from skin and gastrointestinal tract (10.A% is from skin, 6.2% is from gastrointestinal tract). Thus, when calculating the fractional catabolic rate of myosin and actin from the urinary excretion of NT-methylhistidine, these NT-methylhistidine contributions to daily urine output have potential to result in erroreous breakdown rate estimates of skeletal muscle protein and hence NT-methylhistidine contribution from these two tissues should not be overlooked (Nishizawa et al., 1977; Rennie and Millward, 1983; Ballard and Tomas, 1983). 25 Table A. NT-Methylhistidine Content of Actin and Myosin from Different Speciesa * ** Species Muscle Actin Myosin NT-Methylhistidine-—— Rat (growing) Mixed skeletal .78 .88 Rabbit (adult) Hind leg - 1.8 1.3 _ .88 1.63 .86 - Chicken (adult) Mixed skeletal .73-1.05 - Mouse Mixed skeletal - .78 Pigeon Breast — .91 Lobster tail Invertebrate .MA 2.55 Striated ~ 1.8 Cow uterus Smooth .80 . 1.05 Sheep Lateral thigh - 1.7 Cat Flexor hallucis .- 1.3 longus Fowl Pectoralis - 1.5 Cat Soleus .7“ - aAdapted from Haverberg et al., 197A. * Expressed as moles per H1,785 g of protein. ** Expressed as moles per 500,000 g of protein. 26 Huszar et a1. (1983) found a sudden rise in NT-methylhistidine excretion shortly after meat ingestion by man. Since free NT-methylhistidine is excreted rapidly, this rapid rise was most likely due to rapid absorption of soluble NT-methylhistidine from the meat. f. NT-Methylhistidine in Plants or Microorganism Orchardgrass hay and concentrate (composed of corn, barley, bran, rice bran, soybean meal and salt mixture) contained 2.uo and 7.73 mg NT-methylhistidine/kg (mean values of five determinations), respectively, according to Nishizawa et al., 1979. Since the mean intake was 1.58 kg/d for orchardgrass hay and 5.79 kg/d for concentrate, each animal (217 kg, 8-9 months) obtained approximately U9 mg NT-methylhistidine/d from their feed (Nishizawa et al., 1979) and this is a physiologically significant quantity which will influence urinary NT-methylhistidine output. These workers also found that rumen protozoa contained trace amounts of NT—methylhistidine, but NT—methylhistidine was not present in rumen bacteria. Rumen contents contained approximately .131 mg NT—methylhistidine/kg fresh material. Feces contained 1.51 i .1“ mg NT—methylhistidine /kg dry matter which suggested that cattle at 312 kg body weight voided 2.A9 mg NT—methylhistidine/d into feces. From feed intake and fecal excretion of NT—methylhistidine, apparent digestibility of NT-methylhistidine in the feed was calculated to be 98%. Almost 30% of the urinary 27 NT—methylhistidine output was contributed by NT-methylhistidine in the feed if NT—methylhistidine from feed was neither degraded nor metabolized in the digestive tracts of the cattle. More recent studies indicated that there was .21 mg NT-methylhistidine per gram of dried mixed orchard-brome hay and no measurable amount of NT-methylhistidine in corn silage (Wohlt et al., 1982). Previous workers found no NT-methylhistidine in rumen bacterial hydrolysates, free NT-methylhistidine does not occurs in the rumen liquor (Leibholz, 1968), but more recently others found .023 mg NT-methylhistidine per gram of dry rumen bacteria (Wohlt et al., 1982). The limited results indicate that feeds can contribute significant quantities of exogenous NT—methylhistidine to ruminants thus casting doubt on the validity of the NT-methylhistidine procedure. To adequately determine the usefulness of NT-methylhistidine as an index of myofibrillar protein turnover in ruminant animals, definitive studies must be conducted to determine if plants and microbes contian NT-methylhistidine (Wohlt et al., 1982). g. Analysis of NT—Methylhistidine -- Early and Newer Methods The potential of the NT-methylhistidine measurement has been quickly realized in clinical studies (Young et al., 28 1973) but with the obvious importance of lean meat in animal farming practice the technique is certain to have a vital role in agricultural research (Lobley and Harris, 1977). NT-Methylhistidine is usually measured after separation from urinary metabolites on an ion-exchange column of an amino acid analyzer, or by high performance liquid chromatography (Ballard and Tomas, 1983). Haverberg et a1. (197A) used a modification of the method of Spudich and Watt (1971) for purification of actin and consequent isolation and quantitation of NT-methylhistidine in contractile proteins. Haverberg et a1. (1974) described the procedures for isolation of purified actin and myosin from mixed skeletal muscle as well as preparative method for isolation of a concentrated NT-methylhistidine fraction from protein hydrolysates for ion-exchange chromatography. The purity of the preparation was checked by the presence of a single band on both 5 and 10% sodium dodecylsulfate polyacrylamide gels according to the method of Weber and Osborn (1969). Wassner et al. (1980) utilized precolumn derivatization and subsequent high-pressure liquid chromatographic separation of NT-methylhistidine from urine and plasma, using a solvent of 10 mM sodium phosphate (pH 7.5) and an acetonitrile gradient (20-A0% acetonitrile over 15 min at 1.5 ml/min using a concave curve). The fluorescence of NT-methylhistidine decreased slightly as the concentration 29 of acetonitrile was increased and in the range 20-U0% acetonitrile fluorescence was approximately 80% of maximal. This elution can be perfomed isocratically and requires less than 10 min. Both fluorescent and ultraviolent detection may be utilized. Wassner et a1. (1980) also pointed out that this method is at least 103 times more sensitive than conventional ion-exchange chromatography using ninhydrin. Wassner et a1. (1980) demonstrated that the use of this precolumn derivatization method and high- pressure liquid chromatographic separation of NT—methylhistidine has several advantages over current ion-exchange chromatography techniques: First, only a single solvent is required, second, there is no need for column regeneration, and finally each run is several times faster than ion—exchange chromatography. The need for a second reagent pump is also eliminated when using precolumn rather than postcolumn derivatization. The specificity of the fluorescamine derivaties reaction eliminates the problems of coelution of other competing basic amino acid peaks when using ion-exchange chromatography and post—column derivatization. This method is extremely sensitive using fluorescamine detection with the limits of detection less than 600 fmol (600 x 10‘15 mol). Mattews et a1. (1981) utilized a simple, sensitive method for measuring NT—methylhistidine in biological 30 samples using a deuterated internal standard and methane chemical ionization gas chromatography-mass spectrometry. After sample preparation, a single analysis can be completed in 3 min, bringing the total time to 15 min per sample. Nanomole amounts of NT-methylhistidine in urine or plasma sample are determined with a precision of .5%. Picomole amounts are measured with a precision of 10%. Mattews et a1. (1981) pointed out that the primary advantage of the gas chromatographic-mass spectrometry method for measuring urinary NT-methylhistidine is speed of analysis. Changing from ion-exchange chromatography to gas chromatography—mass spectrometry selected ion detection can improve the measurement of a small NT-methylhistidine peak surrounded by other large peaks. The gas chromatography- mass spectrometry assay is ideal for measuring NT-methylhistidine concentration in urine, plasma and muscle samples. Murray et a1. (1981) used a modification of the method of Nakamura and Pisano (1976); a modification of the reaction of fluorescamine with amines, which renders it specific for certain imidazoles. Interference due to histidine and histamine is selectively removed by prior reaction with aldehydes. The concentration of NT-methylhistidine in human urine as determined by this technique correlate well with those determined by ion-exchange chromatography. The technique is rapid (6-7 31 samples/h), is reproducible, requires no prior treatment of the sample, and can be implemented with widely available diverse equipment (Murray et al., 1981). Ballard and Tomas (1983) pointed out that since creatinine in urine is also measured conveniently with a relatively straightforward and rapid technique, the Technicon Auto-Analyzer, is usefull for quantifying the fractional rate of muscle protein breakdown both for clinical assessment of patients and for research purposes. A radioimmunoassay procedure has now been developed for NT—methylhistidine (Bachmann et al., 198u). This should improve speed and specificity of the analytical scheme. MATERIALS AND METHODS The procedures uses were modifications and combinations of several methods. First column chromatography was conducted according to Haverberg et al. (197“) to determine the qualitatively NT—methylhistidine elution volume using pyridine/H20 mobile phase. Second ion exchange chromatography analysis and total analytical system recovery after pyridine chromatography was used to quantify NT-methylhistidine and establish the practical recovery method. Upon successful application of the methodology, feedstuffs analysis of NT—methylhistidine followed. The feed samples were analyzed with and without external addition of NT—methylhistidine. NT—Methylhistidine‘in duodenal digesta from steers, was also determined, to assess the potential of ruminal microbes and bypass feed protein to contribute physiologically significant amounts of NT—methylhistidine toward the urinary excretion. Further ruminal degradation of NT-methylhistidine was evaluated using an in 31339 rumen fermentation. 'Finally, red and white bovine muscles from young and finished cattle were analyzed for NT-methylhistidine to assess muscle type and developmental effects on skeletal muscle NT—methylhistidine content. ‘ 32 33 I. Pyridine-Column Chromatography Procedure A 1.5 x 7.5 cm column of Dowex 50 W -X 8 (strongly cationic) resin of 200-“00 mesh, was washed with 50 ml deionized H2O to desalt the resin and equilibrated with A0 ml .2 M pyridine. These washings were discarded. NT-Methylhistidine elution from the Dowex 50 column with pyridine was evaluated by the following modification of the method of Haverberg et a1. (1979). Two ml 1.0 mM NT-methylhistidine (Sigma Chemical Company) in .01 N HCl diluted in 8 ml .2 M pyridine (final volume 10 ml) or 1 ml 1.0 mM NT—methylhistidine in A ml .2 M pyridine (final volume 5 ml) were applied to columns as samples. Next, 100 ml .2 M pyridine were washed through the column (under N2 pressure) and ten 10 ml fractions of column elutate were collected. This was followed by 125 ml 1 M pyridine elution, and hence 25 fractions of 5 ml were collected. A further 100 m1 1 M pyridine was then used as eluent and 10 tubes of 10 ml were collected. One ml aliquots from each collected fraction were checked for amino acid content by adding 2 m1 ninhydrin solution (Appendix Table A. 1.). The samples were then boiled immediately for 15 min, cooled and visually inspected for dark purple color; the product of a 0 amino acid - ninhydrin reaction. II. NT—Methylhistidine Analysis by Ion Exchange Chromatography and Total Analytical System Recovery Various concentrations and samples of 3U NT-methylhistidine and histidine were subjected to pyridine column chromatography, and the collected samples were analyzed by an amino acid analyzer to determine the pyridine elution pattern and recovery of NT-methylhistidine. Standard amino acid solutions were mixed in three varying combinations and applied to Dowex 50W - X8 columns as follows (Table 6) l) 1 ml 1.0 mM NT-methylhistidine, 1 ml 1.0 mM histidine and 8 ml .2 M pyridine 2) 2 ml 1.0 mM NT-methylhistidine, 1 ml 1.0 mM histidine and 7 ml .2 M pyridine 3) .5 ml 1.0 mM NT-methylhistidine, 2 ml 1.0 mM histidine and 7.5 ml .2 M pyridine The pyridine column chromatography procedure as used for samples above was repeated twice for each of the samples. After discarding the first 100 ml of .2 M pyridine from each column, 60 ml of eluent (1 M pyridine) were collected in a beaker. After air evaporating off the pyridine-H20, 1 m1 aGPA (a-Guanidino Propionic Acid; Pierce Chemical Company) and 9 ml lithium citrate sample buffer (lithium citrate sample dilution buffer: .2 N Li+ (pH 2.20.1 .01 contains 1% thiodiglycol and . % phenol; Pierce Chemical Company) were added to the beaker. The solutions were recovered, filtered through a .2 up memfical membrane filter (Gelman Science Inc., Ann Arbor, MI), and 35 samples were then applied onto the amino acid analyzer. A total of .1 m1 sample was applied to the amino acid analyzer. The visible detector (570 nm) was at a setting of .5 AUFS (Absorbance Units Full Scale), the range for the analysis was 1-20 nmoles. The conditions of NT—methylhistidine analysis were : Column, 26.5 cm by 3.2 mm i.d. containing DC ”A resin (Dionex Co.) with a mobile phase flow at 12 ml/h at 60°C. A four buffer step gradient was used to elute basic amino acids as follows: .25 N Li citrate, pH 2.7, 1 minute; .6 N Li citrate, pH 3.2, 5 minutes; 1.0 N Li citrate, pH A.2, A0 minutes; and 1.3 N Li citrate, pH 5.1, 65 minutes. Postcolumn derivatization was achieved with ninhydrin added at 6 ml per hour. III. Analysis Technique for Feedstuffs and NT—Methylhistidine Recovery Procedure Twelve feed samples, alfalfa (Medicago sativa L.), beet pulp (Beta vulgaris L.), birdsfoot trefoil (Lotus corniculatus L.), corn silage (Zea mays L.), tall fescue (Festuca arundinacea Schreb.), high moisture corn (Zea mays L.), Kentucky bluegrass (Poa pratensis L.), orchardgrass (Dactylis glomerata L.), Italian ryegrass (Lolium multiflorum Lam.), soybean meal (Glycine max Merrill) and timothy (Phleum pratense L.) were analyzed. Except alfalfa, beet pulp, corn silage, high moisture 36 corn, soybean meal, the other 7 feeds were all cut by a competent person on the university farm in Fall 1983. All samples were dry and ground in a Wiley mill through a medium fine screen. Initially crude protein content was analyzed for each sample with micro Kjeldahl digestion followed by NH3 detection using the Nessler (hypochlorite) reaction. Twenty-five mg protein and 50 mg protein samples of each feed were weighed in duplicate for acid hydrolysis. Hydrolysis of feedstuff samples was accomplished with 10 ml 6 N HCl at 121°C for 16 hours in sealed tubes previously flushed with nitrogen gas for 30 seconds. Following hydrolysis and cooling, hydrolysates were filtered through No. 1 filter paper (Whatman Inc, Clifton, NJ.) and washed with deionized H O; the filtered samples 2 were collected into 250 m1 round bottom flasks and evaporated to dryness under vacuum with a rotary , evaporator. The residues were resuspended 10 m1 .1 N HCl. The filtrates were decolorized with a charcoal column (Appendix Table A.2.) as follows: Samples were applied to the column and washed twice with 15 ml deionized H20, and the filtrates (elutates) were evaporated to dryness under vacuum with a rotary evaporator at 500C. The dried hydrolysates in the 250 ml round bottom flasks were then dissolved in 5 ml of .2 M pyridine and 37 applied t0 a Dowex 50V! - ){8 column (1.5 x 7.5 cm; 200-300 mesh, Bio-Rad Laboratories, Richmond, CA.). These column had been prewashed, desalted and equilibrated (50 ml H20; “0 m1 .2 M pyridine) as describes above. The acidic and neutral amino acids were eluted with 100 m1 of .2 M pyridine and the NT-methylhistidine fraction was then eluted with 60 m1 of l M pyridine. Other amino acids remaining on the column were eluted with an additional 125 m1 of l M pyridine; this fraction was discarded. The NT-methylhistidine fraction was evaporated by rotary evaporator (500C water bath) to near dryness and the residue was dissolved in 1 ml lithium citrate sample buffer, filtered through a .2 um filter and then analyzed by ion-exchange chromatography. The NT-methylhistidine content and recovery of externally added (spiked) NT-methylhistidine from alfalfa hay and hydrolysis was determined using the procedures outlined above with 6 various combinations of treatments in duplicate as follows: 1) Samples containing 25 mg protein of hydrolyzed alfalfa hay and mixed with 1 ml .1 mM NT-methylhistidine 2) Samples containing 25 mg protein of hydrolyzed alfalfa hay 3) Samples containing 50 mg protein of hydrolyzed alfalfa hay and mixed with 1 ml .1 mM NT—methylhistidine A) Samples containing 50 mg protein of hydrolyzed alfalfa hay 38 5) Samples containing 100 mg protein of hydrolyzed alfalfa hay The hydrolyzed samples were exposed to charcoal cleanup and pyridine chromatography. 6) 1 ml .1 mM aGPA, 1 ml .1 mM NT—methylhistidine Two further combinations of amino acids were used (7 and 8) to obtain recorder response values to quantitate combinations 1-6. 7) 1 ml .1 mM aGPA, 1 ml .1 mM NT—methylhistidine were mixed, was injected into analyzer directly 8) 1 ml .1 mM aGPA, 1 ml .1 mM NT—methylhistidine were mixed and diluted with 2 ml .01 N HCl and injected on the amino acid analyzer directly. IV. NT—Methylhistidine Analysis of Duodenal Digesta from Steers The duodenal samples were obtained from Dr. William Vernon Rumpler's research samples, which were obtained by the following collection procedure (Rumpler, 1988): At each sampling time a duodenal digesta sample (350 ml) was obtained and frozed until composited. Compositing of duodenal samples involved homogenization of the whole digesta sample in a large blender after thawing. Equal amounts (100 g) of each wet homogenate were then added to a composite sample at the end of the four day collection period. Duodenally fistulated Holstein steers 39 were fed four different diets, as followed: A. 31% DM alfalfa haylage B 31% DM alfalfa haylage plus high moisture corn (HMC) C. “5% DM alfalfa haylage D “5% DM alfalfa haylage plus HMC These four duodenal digesta samples represent each of the four diets from block 1 of a “ x “ latin square. It was not felt to be necessary to analyze all 16 duodenal samples for all “ blocks. The duodenal samples were weighed into hydrolysate tubes and NT-methylhistidine content determined as detailed for the feed samples except recovery studies were not performed. v. Ruminal Degradation of NT—Methylhistidine 12 31339 Ruminal catabolism 1g 11339 of leucine and NT-methylhistidine was determined with rumen fluid collected before and “ hours after feeding. The rumen fluid donor was a rumen fistulated crossbred steer (6“0.9 kg) fed once daily 16 kg corn silage with .69 kg of supplement 8201 (Table 5). Rumen fluid was collected once weekly over a four week period. Two samples were obtained before feeding and two samples four hours after feeding. After collection, the rumen contents wanestrained and squeezed through 2 layers of cheesecloth into a thermos. Approximately 200 m1 of strained rumen fluid (SRF) was collected every time. “0 Table 5. 8201 Supplement Composition Item International Reference No. % Soybean meal 5-0“-60“ 8“. Dical (Ca2POu) 6—01-069 7. Trace mineral salt - “. Limestone (CaCO3) 6-02-632 l. Selenium 90 - 2. Vit Aa - b Vit EC — a Vit A palmitate, 60,000 IU/kg of final supplement. b Vit D3, “,500 IU/kg of final supplement. cVit E, 500 IU/kg of final supplement. “1 The in yitrg_rumen fermentation was initiated by mixing 50 m1 SRF and 50 ml Ohio buffer (prepared according to Appendix Table A. 3.) and subsequently adding Nremethylhistidine and leucine to a final concentration of 1 mM. The SRF—Ohio media mixture was then bubbled with CO2 for 15 minutes and incubated with a Bunsen stopper at 3900 in a water bath. Samples were obtained from the ‘ fermentation at 0, 1, 2, “, and 8 hrs. Samples were deproteinized with .1 volume of 50% sulfosalicylic acid (SSA), stored in ice for 30 min, centrifuged at 15,000 xg for 30 min and the protein free filtrate was ccllected. The filtrates wenadiluted 10 x with .01 N HCl, then 1 m1 sample was mixed with 1 m1 .1 mM aGPA and filtered through a -2 um membrane filter. Leucine and NT—methylhistidine were determined with the amino acid analyzer as outlined above. VI. NT-Methylhistidine Content of Bovine Muscles Four different muscles were taken from three veal calves (38.6 kg, 1 wk of age; 70.9 kg, 17 wks of age; 227.3 kg, 33 wks of age) and three finished steers (518.2 kg, 18 months; 559.1 kg, 18 months; 587.3 kg, 18 months) after slaughter. The muscles removed were longissimusckxsi (LD, white muscle), gluteus medius (GM, white muscle), gluteus profundus (GP, red muscle), and vastus intermedius (VI, red muscle). Muscle tissues were finely sliced; .5 g “2 of each sample was weighed in duplicate for HCl hydrolysis followed the pyridine-column chromatography procedure and the NT-methylhistidine analysis as outlined above. A total of “8 muscle samples were analyzed for NT—methylhistidine content. VII. Statistical Analysis To evaluate the statistical differences among muscles, a one way analysis of various was conducted (Gill, 1978). Bonferroni t statistic was used to compare the,difference of NT-methylhistidine between veal and steer muscles. In addition, it was used to compare the difference of NT—methylhistidine concentration between red and white muscles; if P <.1 the level of statistical significance was reported. P <.05 was determined as being significant. Statistical analysis (ANOVA) was achieved with a program written for the Hewlett Packard 9825A. RESULTS AND DISCUSSION I. Pyridine Column Chromatography Figure 6 shows Dowex 50‘W -X 8 chromatography of NT-methylhistidine, histidine and aGPA with pyridine-water eluents. NT-Methylhistidine was visualized in the tubes 5-8 or in 20—“0 ml elution volume of initial 1 M 125 m1 pyridine fraction., No-ninhydrin—amino acid reaction was' seen in the initial 100 ml .2 M pyridine elution and in the additional 1 M pyridine elution. In the next experiment, 2 ml 1 mM histidine was dissolved in 8 m1 .2 M pyridine and applied to the Dowex column and the pyridine elution was followed as for INT-methylhistidine. Figure 6 also shows histidine was found in the tubes 17—22 or in elution volume 85 to 110 m1 of the 1 M pyridine eluent. _Likewise, 2 ml 1.0 mM aGPA ‘was diSsolved in-8 m1 .2 M pyridine and applied to Dowex column and eluted with pyridine as above. oGPA was found in tube 25 or in elution volume 120 to 125 m1 of 1 M pyridine eluent (Figure 6). II.. Amino Acid Analysis and NT-Methylhistidine Recovery Ion-exchange chromatography was utilized to confirm that the NT-methylhistidine elution volume indicated in “3 ““ Figure 6.8 Dowex 50 W - X8 Chromatography of NT-Methylhistidine, Histidine and aGPA with Pyridine-Water Eluents. 3.5 253». £53m . . HE OCH He 9: 65386 z HVTII. llllll E mmfi 65368 s H IllllYTofioEE . E N. mmH omH OHH mm 0: ON 0 o on poHpo opmmmHoppmn mm: o» pmppm was ochHpmHnHmnpszHz sodem: .m oerHomHeHssoozuez CCH QQCH COH I I moH I ma mo ARV mho>ooom I I cm om oo oo cw oo 2 H CCHCHLHm m I I I I I I I 2 Ho. Hum H H H I I I I I SE H. ooom oeHoHomHeHsecozIwz .s oHooe 6“ .Umpoouoo 902 fl oz m mm. mz.HH :mo mo H .H omCCHMHQ EsmHgm mcpoEHB mH.N mm.w: now :0 m HHHLLmE me @632:me HmmE Gmmnhom poz so.om one so m .ewH soonoHoHoe eoHHoH epHHooH .mmmowoag mm. os.mH oHs so H .H momtoeon mHHHoomo pmpwmowccoho ooz ss.m mom oo H .H mHmooopwo poo mmcwwosHo sxospcog MH.H ss.m mHm mo 3 .H made mom choc owsomHoe gmHm mDZ mw.HH :00 HO H .thflom mmomCHUCSeHm wodpmmm Haw» nmSommm sm.H oo.m mmm mo m .H uses mom oprHm cgoo maz mm.mH mew mo H .mmon mHEpocH mssopm mmmpmeogm Hm. os.mH mmm we H .H oopmHsochoo noooH HHooowo ooowmthm mH.H om.s moo 00 a .H mepmHo> boom oHso comm mm.H sm.mH mom we H .H o>Hcmm owcoHcoz ppreHg Izo M\HOE:I I H I mafipHumHn cHopopm .oz mocomomom osz OHMHpcoHow EopH H>soozez pesto .HpcoprowoocH moosomooog so ocHoHomHsHHgoozIwz pop oHooooa posse .m oHoce 65 For a 300 kg steer consuming 2% of body weight of soybean meal. 300 kg x 2% = 6 kg = 6,000 g 6,000 g x 2.15 nmol/g DM = 12,900 nmol = 12.9 pmol Since normal urine NT-methylhistidine excretion range from l—“ mmol/d (Benner , 1983; McCarthy et al., 1983), the potential contribution from feedstuffs is less than 1.5% of urinary excretion. Nishizawa et a1. (1978) reported that they found significant amounts of NT-methylhistidine in maize, soybean meal, oats, wheat, rice and lucerne meal but did not report the actual values in their paper. Wohlt et a1. (1982) showed that dried mixed orchard-bromegrass hay contained .021 mg NT-methylhistidine/gm but no measurable amounts of NT-methylhistidine were found in corn silage. Using the same assumptions about a 300 kg steer consuming 2% body weight of feed, as above, the Wohlt et a1. (1982) values for orchard—bromegrass hay NT-methylhistidine content would result in an increase of urinary NT-methylhistidine excretion of .72 mmol daily or a 18-72% equivalent of the normal dally NT-methylhistidine excretion (Table 9). A standard chromatogram of 1 : 1 mix, .1 mM NT—methylhistidine and .1 mM aGPA is shown in Figure 13. The chromatogram of a 25 mg protein hydrolysate from timothy is shown in Figure 1“. Figure 15 shows the 66 Table 9. Calculation of Urinary NT—Methylhistidine Excretion in a Steer Fed Dried Mixed Or'ChaI‘d-Br’orz'lesrass Haya 1- Steer, body Weight = 300 kg 2. Feed intake, 2% body weight = 6,000 g 3. NT-Methylhistidine .12 nmol/g dry feed “. 6,000 g x .12 nmole/g dry feed = .72 mmol/Q1 5. Normal urinary NT-methylhistidine excretion = 1-“ mmol per day 6. Contribution from orchard-bromegrass = 18-72 % of normal urinary Nr-methylhistidine excretion Based on values from Wohlt et a1. (1982). 67 O C O a ‘ 0 g ABSORBANCE at 570 nm .10 .05 68 Figure 1“. 1 , Legend 1 jNT—Methylhistidine l ' L ‘ 3 2 aGPA ‘ 38 an 56 68 80 Elution Time (min) 69 timothy with a external spike of 1 ml of .1 mM NT-methylhistidine. Further the NT-methylhistidine was also not appearing in basic amino acid chromatograms at another retention time (Figure 15) confirming that NT-methylhistidine was not missed. It can be noted that .55 nmol/g DM of NT-methylhistidine was detected in the original feedstuff (Table 8). According to the calculation of these two experimental data, Wohlt et a1. (1981) result .72 mmol NT-methylhistidine per day for 300 kg steer consuming 2% body weight orchard-bromegrass is 56 times higher than the result of present study 12.9 umol NT-methylhistidine per day for 300 kg steer consuming 2% body weight of soybean meal, as well as 218 times higher than feeding 300 kg steer of timothy hay. Hence it is clear that the feedstuff of present study does not contribute significant amounts of NT-methylhistidine. IV. NT-Methylhistidine Content in Duodenal Digesta of, Steers NT-Methylhistidine was determined in four composite duodenal samples from block 1 of a “ x “ latin square digesta passage study (Rumpler, 198“). The results of these analyses are presented in Table 10. The duodenal samples contained 16-2“ % crude protein and NT-methylhistidine was detected only for the “5% DM alfalfa haylage-high moisture corn diet, the 70 Figure 15. Chromatogram of External "Spike" of NT—Methylhistidine 25 mg Protein Timothy Hay Hydrolysate. ABSORBANCE at 570 nm .50 .“5 .“0 .35 .30 .25 .20 .15 .10 .05 71 ““ 56 68 7“ 80 Elution Time (min) Figure 15. Legend 1 2 NT-Methylhistidine aGPA 72 .pcoooooo cos u as o .mHmmo spouse spa m . oss + m.H m.mm o.m ompHsps msHmsHp so Hm: s ooz m.mm m.m ompHsps osHmsHp so Hm: m . oss + ooz m.mH m.m ompHsps psHpsHm so sHm m ooz _m.mH o.m ommHsps moHpsHm so sHm H I M\oHoE: I & ochHomHsHssposIsz cHoooss costs cowowcHz coHoccHosoo poHo oHoEpm eacoooso mmsmopm Eosm momoch Hccooooc cH oooocoo cHoooss ooowo pep comoonz .ocHoHomHsHssoosIsz .OH oHoms 73 NT-methylhistidine was below detection limits (.2 nmol) for the other three digesta samples (Table 10). The daily dry matter flow for steers fed the “5% DM alfalfa—HMC diet was 3,269 g and hence the upper level of NT-methylhistidine flow was 3.9 nmol/d (Rumpler, 198“; Appendix Table A. 5.). This value is far below the daily urinary excretion of NT—methylhistidine. The conclusion can thus be reached that, at least for alfalfa haylage-HMC diets, neither ruminal microbes nor the feeds contribute a physiological significant amount of NT-methylhistidine to cause increased urinary NT—methylhistidine excretion which in turn result in overestimated fractional breakdown rates of skeletal muscle (McCarthy et al., 1983). The duodenal digesta chromatogram confirms that no significant amount of NT—methylhistidine is present (Figure 16) and internal standard aGPA is appeared in the same retention time as above. V. NT-Methylhistidine Degradation by Ruminal Microorganism Ig’Vitgo Rumen fluid obtained before and “ hours after feeding and NT-methylhistidine and leucine (at 1 mM) were incubated with rumen fluid and a buffer for 0 and 8 hours. Analysis for both amino acids was aided by the fact that the rumen fluid alone (diluted 1:20) contained no detectable amino acids (Hungate, 1966; Bergen, unpublished 7“ Figure 16. Chromatogram of Duodenal Digesta Sample. ABSORBANCE at 570 nm 75 .“5 % .15 '101I- .05 :J\»\__,( Figure 16. Legend \J 1 aGPA I I I I l ““ 56 68 7“ 80 Elution Time (min) 76 observation) and hence dilute SRF blanks were not run. Amino acid degradation was calculated from the formula: Initial (0 hour) Conc. - Final (8 hour) Conc. Initial (0 hour) Conc. x 100% Results of this experiment are given in Table 11. After an 8 hour incubation, leucine was not detected on the chromatograms and its degradability was therefore 100% (Table 11). The rate of degradation for NT—methylhistidine was slight and not influenced by the source of rumen inocula. These results indicate that if a feedstuff contains high levels of NT-methylhistidine this amino acid. would escape ruminal catabolism 1Q 1139, be absorbed from the small intestine and could thus lead to errors in the calculation of FBR based on urinary NT-methylhistidine excretion. Previous workers reported that free NT-methylhistidine probably does not occur in the rumen liquor (Leibholz, 1965) or in hydrolysed rumen bacteria (Bergen and Potter, 1971).. Our preliminary work with blank-diluted rumen liquor also detected no NT-methylhistidine. This study shows that NT-methylhistidine degradation in rumen fluid is only 1“.5%, while typical protein bound amino acids, like leucine,-not surprisingly are completely degraded under the same incubation conditions. 77 Table 11. The Degradation of BCAA-Leucine and NT-Methylhistidine After Incubation for 8 Hours of Rumen Fluid Collected Before Feeding and “ Hours After Feedinga Degradation Amino ACid Source of Rumen Fluid Before Feeding “ Hours Post Feeding g. NT—Methylhistidine 1“.6 1“.3 Leucine 100 100 a Each value is the mean of two separate in vitro fermentations. 78 VI. NT-Methylhistidine in Bovine Muscles The results for this experiment are presented by age of cattle and across muscle types (red or white) in Table 12 and strictly as a mean concentration of the four muscles analyzed for each age group in Table 13. The LD and GM muscles (white) of calves and slaughter (beef) steers had an overall mean NT-methylhistidine content of .68 nmol/g fresh tissue. The NT-methylhistidine content of GP muscle of the veal was significantly lower (P< .05) than the value for the' slaughter steers (Table 12), but for the other red muscle (VI), veal only showed a marked numerically lower, but nonsignificant, Nt-methylhistidine content than that observed in steers. When all four muscle samples were grouped for the young and finished slaughter steers, the mean total muscle NT-methylhistidine content tended to be significantly lower in the young than the finished steers, however the samples from the 1 week old veal calf gave average NT-methylhistidine value near typical of the older cattle (Table 13). Table 1“ shows that NT-methylhistidine concentration in adult human skeletal muscle has been established at. approximately “.2 pmol per g mixed muscle protein and 3.2 nmol/g protein for infants, but probably considerably less in the fetus due to the lack of methylation of fetal 79 .HosmH .tHoHspmppsm ccm gammy mom mH msoopm Eopm mHomSE no coHpHmOQEoo mumpcoopoo cHoposd ommso>< ** .Amc. vmv HCQMHU mpoHpomLCQSm pcopomme csz mzos cngHz mcmmz no .58 pcm wasps ppm mpus> * :3 so. ms. mm. mm. mm.m mm.m mszoELCHCH maumm> 33 mo. m me. n::. mH. as:.m oom.m mapcssoso msmpsHu pom co. Hs. mm. cm. mm.m mm.m Azcv msHpoE mSCHSHo mo. ms. _sm. mm. sm.mw mH.m HQHVHmsoo mseHmmecoH 2mm I**o:mme nmosm m\ HoE: I 2mm IxscHopoLQ. M\ Hoe: I much cogchHg ucsow poanch. wcsos pose oHowsz *pcopcoo mchHpmH£H>£pozIsz whooom cosmHng con ucaos 1: ocoocoo ocHoHomHsHsfiosIez 9:. .mH oHops .zmm pom mcmoE ppm mosHm> ** _ . H. v m * .Hmo. vmv Homqu mpaHHomHCQSm ucmsompHp cpHs :ESHoo chsz mammz n m mo. MH. smm mo. os.m m.som msocos oH ems. m Houm H.omm nachos oH s. oo.m m.mHm pounce mH #63 * .m comm O 2. so. Hm. smm *sm. soo.m m.smm msocs mm oom. oos.m o.os asap: sH mo. mm.m o.om sods H Hmo> I ozmmHu nmosm w\ Hosa. I :Hmposm w\HoE: wal *socoocoo ooHoHomHsHsgoosIez osmHos soom ows oHoooo so moms ocosoooHo om ocousoo ocHoHomHsHsooosIez one .MH oHooe .msmH .oscsz pcm mcsow Eons pouomp< 81 m HmpmoosopCH pmoq m.s .mooms .mHmesoo choocs s oHoo< m.H . msHECCQOLuwmw H uH5p¢ m.m mmomm m pcmmcH - Amxooz onlomv s.H moooHspmsc potz zH msuom CHwPOLQ &\HOE: ocHoHomHsHssoosIez boss oHopos .oz oHoeom cosss mcmEsm Eons moHomsz CH mcHoposm post so ucopcoo ochHpmHannp62Isz .zH oHnme 82 myosin and to the lower proportion of myofibrillar to total protein in muscle at this stage than in later neonatal life (Young & Munro, 1978). Table 15 shows that NT-methylhistidine content of myosin in rabbit; NT-methylhistidine was not detected in the mixed skeletal muscle of 28 day fetus rabbit. In the mixed skeletal muscle of 23 day old rabbit 1.15-1.22 residues of NT-methylhistidine per mole myosin was detected. An average of 1.5 NT—methylhistidine residues per mol myosin was found in adult rabbit muscle. According to the present study, finishing steers at 18 months of age had an average of 3.5 nmol NT—methylhistidine per g protein, while an average of 2.8 nmol NT-methylhistidine per g protein was found in 1-33 weeks old veal calves. Hence, the data from the present study agree with the results of Trayer (1968) for rabbits, and the observation of Young & Munro (1978) for humans. Adult animals usually have higher concentration of NT—methylhistidine than young animals. As expected, red and white skeletal muscle samples contained significant amounts of NT—methvlhistidine (Figure 17). During muscle myofibrillar protein (actin or myosin) turnover, the NT-methylhistidine is released and will not be metabolized or be reutilized in protein synthesis. The sole metabolic fate of NT-methylhistidine 83 .mmmH .smsmse Eons popowp< .m mm.H I mH.H sop mm HpooHoxm post HHz hoops soc mm HoooHosm post mm.H oHsoc oscos mo.H oHsop Hosoo mssHouncoH. oHoopm I HoE\m:pHmoL I ocHoHopHsHssoostz HoeHcp oo oma oHomss HmeHc< meHmoss so ocoocoo ocHoHomHsznoosIez .mH oHops 8“ Figure 17. Chromatogram of Bovine Muscle Sample. ABSORBANCE at 570 nm 85 50 62 7“ 80 Elution Time (min) Figure 17. Legend 1 NT-Methylhistidine 2 aGPA 86 is then to be excreted in urine (Young and Munro, 1978). In some species, the NT-methylhistidine combines with B ahnune to form balenine, a histidine dipeptide, and the NT-methylhistidine is not quantitatively excreted immediately in the urine (Harris, 1977); however, ultimately all NT-methylhistidine must be excreted as this compound and is thus not metabolized. This is in contrast to e-N-Methyl Lysine (trimethyl lysine) which is the precursor of “-trimethylaminobutryate for carnitine synthesis and can thus not be used as a muscle protein turnover marker (Bergen & Potter, 1971). Earlier workers (Leibholz, 1968) appeared to have confused N Methyl Lysine with NT-methylhistidine on their Chromatograms of sheep plasma. This is especially interesting in that most NT-methylhistidine is found as balenine in sheep plasma, but sheep have very high plasma N Methyl Lysine and tissue carnitine levels.. Generally dietary NT—methylhistidine intake is of no physiological significance in ruminants. Under circumstance where the dietary intake were to be significant, however, it would be expected that NT-methylhistidine would ostensiyely escape ruminal catabolism, be absorbed in the small intestine, and excreted in the urine thereby causing an error in the estimation of muscle protein turnover rate. CONCLUSION This thesis work was initiated to determine the role of typical feeds and the ruminal fermentation on NT-methylhistidine metabolism in ruminants. It is clear, from this study, that the NT-methylhistidine content of feedstuffs commonly fed to ruminants is very low. Of the twelve common feedstuffs chosen for this research, soybean meal NT-methylhistidine content 2.15 nmol/g was the highest level, but the actual intake of NT-methylhistidine from soybean meal is negligible and hence it can be concluded that feedstuffs are not a source of urinary NT-methylhistidine. This same conclusion can also be drawn for rumen fluid and duodenal digesta. The primary source of urinary NT-methylhistidine is skeletal muscle and the amino acid arises during protein turnover. Based on the ig_yltgg ruminal fermentation studies, NT—methylhistidine is quite resistant to microbial catabolism and would "bypass" the rumen if fed in A significant quantities. This thesis finally details some convenient procedures to quantitate NT-methylhistidine in animal tissue and feed samples. 87 APPENDICES APPENDIX TABLE A. 1. Ninhydrin Solution Preparation Combine 750 ml methyl cellusolve (Ethylene Glycol Monomethyl Ether; Pierce Chemical Company) 250 m1 NaAc concentration, pH 5.51 (Pierce Chemical Company), 20 g ninhydrin, and 3.5 ml Triton x 100 and bubble with N2 gas while stirring. Continue to bubble for 30 min, then add .“5 g SnCl2 and bubble with N2 gas at least 3 further hours. 88 APPENDIX TABLE A. 2. Charcoal Column Preparation Procedure Glass columns, 7 mm i.d., were prepared with glass wool plugs and .5 cm filter aid (ash-free analytical filter pulp, Schleicher & Schnell, Inc., Keene, N.H. 03“31) on top of plug. Slurry a mix of .5 g filter aid and .5 g charcoal and apply to the column. Continue by adding additional .5 cm filter aid on top of filter aid charcoal mix. Finally, wash columns as follows: 2 ml “ N HCl; 2 x with 5 ml 20% EtOH : H2O (V/V); and then with 5 ml of H20. Use a little N2 gas pressure (.1“ -.22 kgs/cm2) for good flows. 89 APPENDIX TABLE A. 3. Composition of Ohio Media Item Concentration Amount Na2CO3 .2 g/ml 10 ml Biotin 10 ug/ml 10 ml Para-Amino Benzoic Acid 100 pg/ml 2.5 m1 (PABA) H20 “5 ml FeCl3 2.6“ mg/ml 7.5 m1 CaC12.2H2O 5.29 mg/ml (FeCl3 and CaCl .2H2O are n one solution) H2O 7.5 ml Mineral Mix "O"a 175 m1 Add deionized water to 875 m1 To stablize the pH bubbled with CO 2 gas 1-2 h. ‘Final pH adjustments to 6.7 are made with .1 N H280“ or .1 N Na2C03. aMineral Mix "0" composition is in Appendix Table A. “. 90 APPENDIX TABLE A. “. Composition of Mineral Mix "0" Item Concentration Na2HPOu 5.65 g/l KCl 2.15 g/l MgSOu.7H2O .582 g/l NaZSOu 0750 g/l 91 APPENDIX TABLE A. 5. 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Lack of in vitro binding of 3 -methy1histidine to transfer RNA by amino-acyl ligases from skeletal muscle. Biochim. Biophys. Acta 199:297. Young, Vernon R., Sunney D. Alexis., B. Suren Baliga., Hamish N. Munro., Wolfgang Muecke. 1972. Metabolism of administered 3-methylhistidine. Lack of muscle transfer ribonucleic acid charging and quantitative excretion as 3-methylhistidine and its N-acetyl derivative. J. Biol. Chem. 2“7:3592. Young, Vernon R., L.N. Haberberg., C. Bilmazes and H.N. Munro. 1973. Potential use of 3-methylhistidine excretion as an index of progressive reduction in muscle protein catabolism during starvation. Metabolism 22:1“29. Young, Vernon R. and Hamish N. Munro. 1978. NT-Methylhistidine (3-methylhistidine) and muscle protein turnover: an overview. Fed. Proc. 37:2291. 3 1293 "'III"!!!IIIIIILIIIIFIIIIIIIEIIIIEs