Scientific Article

An evaluation of plasma homocysteine in the assessment of vitamin B12

status of pasture-fed sheep

JM Furlong* † , JR Sedcole* and AR Sykes*§

Abstract

AIM: To assess the diagnostic potential of concentrations of homocysteine (Hcy) in plasma in

relation to those of methylmalonic acid (MMA) and vitamin B12, as predictors of responsiveness of
young sheep to supplementation with vitamin B12.

METHODS: Eighty-two ewes grazing ryegrass-white clover pasture were used, 39 of which had
been supplemented with a Co bullet and 43 unsupplemented. Thirty days after commencement of
parturition their lambs (n=53 and 59, respectively) were randomly allocated into one of two
treatments, in a 2 x 2 factorial design. Half of the lambs from each group of ewes received an
injection of vitamin B12, while the remainder were controls. The trial commenced 31 October 2001
(Day 0), and continued until 01 May 2002 (Day 182). All lambs were weighed, and blood samples
taken from 16 identified animals from each treatment group, at approximately monthly intervals.
Changes in concentrations of Hcy, vitamin B12 and MMA in plasma, and liveweight gain (LWG) of
the treatment groups were evaluated during the suckling (Days 0–89) and post-weaning (Days 90–
182) periods.

RESULTS: Mean LWG was 40% greater in supplemented than in unsupplemented lambs. The
concentrations of vitamin B12 and MMA in plasma in the unsupplemented lambs were in the
deficient reference ranges of <170 pmol/L and >16 µmol/L, respectively. Mean monthly
concentrations of Hcy in plasma ranged from 1.5 to 4.5 µmol/L but showed no pattern of response
to vitamin B12 deficiency or supplementation.

CONCLUSIONS: Measurement of the concentration of Hcy in plasma as a metabolic indicator of

reduced methylation capability of sheep on typical pastures in New Zealand appeared to have little
value in detection of vitamin B12 responsiveness, and was less sensitive than the concentration of
the vitamin itself or the indicator of adenosyl-cobalamin deficiency, MMA, in plasma. The
possibility that concentrations of Hcy in plasma remain low due to re-methylation of Hcy to
methionine via the alternative betaine-choline rather than the vitamin B12-dependent terahydrofolate
metabolic pathway is rejected, but the possibility is raised that high rates of trans-sulphuration of
Hcy to cysteine in the gastrointestinal tract of grazing sheep could be responsible.

CLINICAL RELEVANCE: The propionate-succinate pathway appears to be the first rate- limiting
pathway in vitamin B12 deficiency, and the product of disruption of this pathway, increased MMA,
* * Department of Agricultural Sciences, PO Box 84, Lincoln University, Lincoln 7647, Canterbury, New Zealand.
† † Current address: Waste Solutions, PO Box 997, Dunedin 9054, New Zealand.
§ § Author for correspondence. Email: sykes@lincoln.ac.nz
is the most reliable indicator of metabolic abnormality in predicting responsiveness to
supplementation.

KEY WORDS: Ewes, lambs, homocysteine, cobalt, methylmalonic acid, vitamin B12, liveweight
gain

DM Dry matter
Hcy Homocysteine
HPLC High-performance liquid chromatography/chromatograph
LWG Liveweight gain
MMA Methylmalonic acid

Introduction

The ruminant animal is solely dependent upon the adequacy of Co intake for synthesis of its
requirement for vitamin B12 (cobalamin). A deficiency can lead to symptoms that include reduction
in appetite, reduced or even negative growth, anaemia, and ocular discharge (Underwood and Suttle
1999).

Vitamin B12 comprises two distinct cobalamins with specific co-enzyme function, namely adenosyl-
and methyl-cobalamin. Adenosyl-cobalamin is co-factor to the co-enzyme for L-methylmalonyl-
Co-A mutase, that catalyses the isomerisation of methylmalonyl Co-A to succinyl Co-A (see Figure
1). This is an essential reaction in the conversion of the odd-chained fatty acid propionate, a product
of fermentation of carbohydrate in the rumen, to an even-chained fatty acid, succinate, prior to
gluconeogenesis or its oxidation to supply energy. Failure of isomerisation leads to the formation of
MMA, and its accumulation in tissues. Methyl-cobalamin is the co-enzyme for methionine synthase
in the exchange of methyl groups between methyl-tetrahydrofolate and Hcy to produce methionine
which, after conversion to S-adenosyl methionine , can donate methyl groups before recycling to
Hcy, via adenosyl-homocysteine, for re-methylation. Methylation reactions are vital for the
synthesis of many key metabolites, such as phospholipids, choline, creatine and carnitine, as well as
in the regulation of DNA activity and oncogene status (Lobley et al.1996).

The diagnosis of vitamin B12-responsiveness from concentrations of the vitamin in plasma is

problematic as concentrations in the blood may not truly reflect the deficiency at a cellular level.
This is recognised in the establishment of wide reference ranges and probability values to predict
production responses to supplementation (Clark et al. 1985). Measurement of the concentration of
MMA in plasma is now seen as a more reliable, though more expensive, diagnostic marker of ovine
responsiveness to vitamin B12 supplementation (O’Harte et al. 1989; Kennedy et al. 1991;
McMurray et al. 1985; Gruner et al. 2004abc).

The concentration of Hcy in plasma is used in diagnosis of vitamin B12 deficiency in human
medicine though its specificity requires the elimination of genetic abnormalities, such as
cystathionine β-synthase deficiency, and renal disease. The concentrations of MMA and Hcy in
plasma in humans without renal disease have been shown to be very sensitive, and provided a
specific diagnosis of vitamin B12 deficiency (Stabler et al. 1996). Elevated concentrations of Hcy
were also observed in vitamin B12-deficient pigs (Stangl et al. 2000a). However, it is unclear
whether vitamin B12 deficiency in sheep results primarily from failure of the propionate-succinate
pathway or the Hcy re-methylation pathway.

Few studies have examined the effects of vitamin B12 deficiency on concentrations of Hcy in plasma
in sheep. A significant elevation in concentrations of Hcy in plasma was reported in lambs kept
indoors, and offered a barley-based diet with extremely low Co concentration; concentrations of
Hcy in plasma rose above 70 µmol/L in association with vitamin B12 concentrations below 220
pmol/L, indicating a decreased capacity to methylate Hcy (Kennedy et al. 1992). Conversely, only
relatively small elevations in concentrations of Hcy compared with those of MMA, were observed
in the plasma of twin lambs grazing Co-deficient pasture (Vellema et al. 1999).

The aim of this study was to determine the value of concentrations of Hcy in comparison with those
of MMA and vitamin B12 in plasma in the diagnosis of vitamin B12 deficiency in grazing sheep.

Materials and methods

The study commenced on 31 October 2001 and concluded on 01 May 2002 (viz Days 0–182), and
was conducted on a property located in Southland, New Zealand (45°50’ latitude south, 168°50’
longitude east), on Mataura silt soil, and identified by the local veterinarian as Co-deficient. The
pastures were traditional perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens
L.).

Eighty-two pregnant 3-year-old Romney ewes from the same flock were used. Of these, 39 had
been supplemented in a previous trial (Gruner et al. 2004a), on Day –330 relative to Day 0 of the
present study, with a 10-g Co bullet (PermaCo; Schering-Plough Animal Health Ltd, Upper Hutt,
NZ) containing 2 g Co as Co3O4. The remaining 43 unsupplemented ewes were controls. The mob
was grazed together as one.

On Day 0 (30 days after the commencement of lambing), the lambs from each group of ewes were
allocated randomly into two groups, one of which remained unsupplemented, and served as
controls, while the other received an I/M injection of 3 mg microencapsulated cobalamin
(SMARTShot B12; Stockguard NZ Ltd, Hamilton, NZ; 1 ml contains 6 mg vitamin B12). This
resulted in a 2 x 2 factorial design, with groups being designated unsupplemented ewe-
unsupplemented lamb (n=29), unsupplemented ewe-supplemented lamb (n=30), supplemented ewe-
unsupplemented lamb (n=26), and supplemented ewe-supplemented lamb (n=27). All the lambs
were weighed, and blood samples obtained from 16 identified animals from each group, at
approximately monthly intervals. At the same time, lambs were treated with a benzimidazole
anthelmintic, as part of the routine farm practice. The lambs were weaned on Day 89, and were
always grazed as a single group. All procedures were carried out under the authority of the Lincoln
University Committee on Ethics of Animal Experimentation, Lincoln, New Zealand.

Sampling procedure and analyses

A strict sampling protocol was adhered to in order to preserve the integrity of samples. Animals
were yarded, and 10 ml of blood collected within 15 minutes by jugular venepuncture, into sodium
heparin vacutainers (140 USP Na heparin/10 ml; Baxter Health Care, Christchurch, NZ), and
immediately placed on ice. The lambs were then weighed and, on Day 0 supplemented groups of
lambs were treated with vitamin B12. The samples were then transported to Lincoln University at
4°C, and plasma separated by centrifugation the following day, at 3,000 rpm for 10 minutes, and
stored at –20°C. Plucked herbage samples were taken on all occasions, by stratified random
sampling from the paddock the animals were grazing, and analysed for Co content.

Total Hcy analysis was carried out using high-performance liquid chromatography (HPLC), with
detection of fluorescence using a modification of the methods described by Araki and Sako (1987)
and Jacobsen et al. (1994), and described in detail by Furlong (2005). Briefly, the release of thiol
was achieved by incubation for 30 minutes in tris-(2-cyanoethyl) phosphine in dimethyl formamide
prior to de-proteinisation. Derivatisation of fluorescence was achieved by incubation for 5 minutes
at 50°C with 7-fluoro-2, 1,3-benzoxadiazole-4 sulphonamide (1 mg/ml) in borate buffer in an
alkaline environment, and immediately placed on ice. Filtered samples were separated and
quantified on an Agilent 1100 Series HPLC (Waldbronn, Germany), using a Phenomenex C18 Luna
(Torrance CA, USA) reversed-phase column. An isocratic system used Eluents A (0.1 M acetic
acid-0.1 M sodium acetate at 600:1,500 v/v, containing 20 ml methanol, pH 4.35) and B (methanol)
for 20 minutes at 85:15 (v/v) at 29°C, and fluorescent activities were measured at 385 and 515 nm.
All standards were prepared in pooled plasma, and cysteamine (as hydrochloride) was added to the
standards and unknowns as the internal standard.

A two-point internal standard method was used for analysis of a sample from a plasma pool (Vester
and Rasmussen 1991) and also a pool spiked with 50 µmol/L Hcy. The slope of the linear
regression line was determined, in which x = the Hcy added, and y = the ratio of the peak area of
Hcy to that of the internal standard, cysteamine. The concentration of Hcy in the samples was then
determined by dividing the ratio between the area of the Hcy peak and the cysteamine peak by the
slope of the linear regression line. The intra- and inter-assay coefficients of variation were 6.44%
(2.48 (SE 0.11) µmol/L) and 6.29% (3.24 (SE 0.11) µmol/L), respectively. As an additional check
on the specificity and accuracy of the assay, random samples from 10 sheep were analysed using an
independent HPLC method based on a fluorescence polarisation immunoassay on an IM-x system
(Abbot Laboratories, Markham, Ontario, Canada), at Canterbury Health Laboratories (Christchurch,
NZ). Mean values were 3.0 and 2.8 µmol Hcy/L for the two assay systems, with intercept, slope and
correlation coefficients of –0.17, 0.963 and 0.96, respectively.

Plasma (~1 ml) was analysed for vitamin B12 content by Labnet Invermay, Mosgiel, New Zealand,
using a radio-isotope dilution assay (RIDA) based on the method described by Green et al. (1974).
The concentration of MMA in plasma was analysed using gas chromatography, using a
modification of the method described by McMurray et al. (1986). The concentration of Co in
pasture was determined using graphite furnace atomic absorption spectrophotometry, following
nitric-perchloric acid digestion (Simmons 1973).

Statistical analysis

Statistical analysis was carried out using GenStat Release 7.2 software (Lawes Agricultural Trust
2004, Rothamstead, UK), as a 2 x 2 factorial design, with factors being supplementation of ewes
and lambs and time, where appropriate. The experimental data were analysed for repeated measures
using restricted measures likelihood, with estimates of missing values.

Results

Concentrations of Co in pasture during the experimental period were variable, with values
decreasing from 120 on Day 0 to 60 µg/g dry matter (DM) at Day 28, before increasing to 210
µg/kg DM at weaning (Day 89), followed by a decline to 50 µg/kg DM at Day 126, before returning
to 210 µg/kg DM by Day 182.

Mean liveweights of the lambs are shown in Figure 2a. There was a highly significant treatment of
lambs-by-time interaction (p<0.001), reflecting a greater LWG in supplemented than in
unsupplemented lambs, that resulted in supplemented lambs being heavier than their
unsupplemented contemporaries, regardless of treatment of the ewes from Day 126 of the trial.

Mean concentrations of vitamin B12 in plasma are shown in Figure 2b. There were treatment of
ewes-by-time (p<0.05) and treatment of lambs-by-time (p<0.001) interactions, reflecting greater
initial values and responses to supplementation in lambs suckling supplemented ewes, and a gradual
convergence of concentrations in all groups subsequently. No difference between treatment groups
was seen from Day 57 onwards, with vitamin B12 concentrations falling steadily to ≤200 pmol/L by
Day 126.
The mean concentrations of MMA in plasma are shown in Figure 2c. There was a treatment of
ewes-by-treatment of lambs-by-time interaction (p<0.001), reflecting a greater increase in
concentrations of MMA in plasma in unsupplemented than in supplemented lambs, and greatest
values seen in unsupplemented lambs from unsupplemented ewes before weaning (42.5 µmol/L on
Day 56) but in unsupplemented lambs from supplemented ewes after weaning (73.3 µmol/L on Day
150).

The mean concentrations of Hcy in plasma are shown in Figure 2d. There was a treatment of ewes-
by-treatment of lambs-by-time interaction (p<0.001), due to higher concentrations of Hcy in lambs
from supplemented ewes on Day 0 and a subsequent lack of consistent difference between groups.
Mean concentrations remained within the narrow range of 1.5–3.0 µmol/L after Day 60.

Discussion

The magnitude of the response in LWG of the lambs to supplementation with vitamin B12, with a
difference in liveweight of 10 kg at the end of the trial, confirmed a severe Co deficiency and an
ideal situation in which to test the hypothesis that the concentration of Hcy in plasma may have
value in the diagnosis of vitamin B12/Co deficiency. There was confirmation of deficiency in
metabolic parameters. Average mean concentrations of vitamin B12 in plasma in the
unsupplemented lambs from unsupplemented ewes (142 pmol/L) and unsupplemented lambs from
supplemented ewes (148 pmol/L) were well below the suggested reference ranges for vitamin B12
deficiency of <230 pmol/L plasma (Suttle and Underwood 1999; Gruner et al. 2004c) on 5/7
monthly samplings. Furthermore, concentrations of MMA in plasma from those lambs were
elevated for the majority of the study into the deficient range suggested by Gruner et al. (2004c) of
>13 µmol/L, and well above the reference criterion suggested by O’Harte et al. (1989) of 5 µmol/L.
By comparison, concentrations of Co in pasture, which fluctuated above and below the critical
concentration of 70 µg/kg DM (Underwood and Suttle 1999), would have been difficult to interpret
alone. Moreover, variation in concentrations of MMA in plasma from unsupplemented lambs
suggests that the degree of deficiency did fluctuate throughout the study, a feature which was not
detected in concentrations of vitamin B12 in plasma, possibly due to the very rapid turnover of the
vitamin in plasma (Ludemann et al. 2005).

In light of the severity and duration of the deficiency, it was surprising that an increase in
concentrations of Hcy in the plasma of lambs was not observed. A 6-fold increase in concentrations
of Hcy was detected by Kennedy et al. (1992) 150 days after commencement of feeding lambs a
barley-based diet with a very low concentration of Co (4.2 µg Co/kg DM), that in a previous trial
had resulted in concentrations of MMA in plasma of 65 µmol/L after 140 days (Kennedy et al.
1990), similar concentrations to those measured in the ewes and lambs on Day 156 in the study
presented here (Figure 2c). In both of the studies by Kennedy et al. (1990, 1992) and the current
study , concentrations of vitamin B12 in plasma were consistently below 200 pmol/L. A fluctuating
vitamin B12 status in the lambs in the current study, which may be anticipated due to the fluctuating
concentrations of Co in herbage and of MMA in the plasma of unsupplemented lambs, could be
responsible. If this was the case, it suggests that, of the two vitamin B12-dependent enzymes,
methylmalonyl-Co-A mutase, involved in propionate metabolism, was more sensitive to deficiency
than methionine synthetase, involved in vital methylation reactions. Conversely, in one of the few
studies in which concentrations of MMA and Hcy in plasma have been measured concurrently
during Co deficiency, concurrent increased concentrations were observed in housed cattle when
offered corn silage concentrate diets with Co concentrations below 160 µg/kg DM (Stangl et al.
2000b). The lack of change in concentrations of Hcy in plasma in lambs in the present study, given
the severity of the deficiency, was therefore very surprising.

The actual mean concentrations of Hcy in plasma observed in lambs with adequate vitamin B12
status in the present study (range 1.5–3 µmol/L) were lower than observations in the literature in
comparable animals on whole barley, of 10 µmol/L (Kennedy et al. 1992) and 38 µmol/L (Kennedy
et al. 1994), hay concentrate mixtures (6–11 µmol/L; Wallace et al. 2006), or pasture (9 µmol/L;
Vellema et al. 1999). Continuing metabolism of red blood cells can result in increases in
concentrations of Hcy (Ubbink et al. 1992). A preliminary trial (data not shown) established that
Hcy in blood collected into sodium- or lithium-heparin was stable at room temperature for 48 hours.
The added precaution of placing samples on ice, and rapid centrifugation, provided an added
safeguard against deterioration of the samples, and falsely high estimates.

In mammals, re-methylation of Hcy to methionine occurs via either the methyl-cobalamin route or
from choline-betaine (Figure 1), a reaction which is not vitamin B12-dependent (Finkelstein et al.
1988). Reduction in the rate of re-methylation results in accumulation of Hcy in plasma. The lack of
accumulation of Hcy in plasma in the lambs in the study presented here may reflect use of the
alternative choline-betaine route but this seems unlikely, for the following reasons. Tissue enzyme
studies (Xue and Snoswell 1985ab) have suggested that sheep are much more dependent than rats
for re-methylation of Hcy by the tetra-hydrofolate-homocysteine transferase route than the betaine-
homocysteine methyl transferase route, which is consistent with the almost complete degradation of
the major dietary sources of the methyl groups choline and betaine by ruminal microorganisms
(Mitchell et al. 1979; Neill et al. 1979). In fact, ruminants are very dependent on the conservation
of choline by extra-hepatic cycling and de-novo biosynthesis from phosphatidyl ethanolamine,
using stepwise re-methylation from methionine (Neill et al. 1979). Moreover, recent feeding trials
have shown milk production responses to supplementation with rumen-protected choline in dairy
cows offered diets with a low supply of methionine (Davidson et al. 2008), indicating the
precariousness of the capacity for methylation in ruminants.
The occurrence of fatty liver syndrome in Co-deficient animals has been reported in grain-fed
(Kennedy et al. 1997) and pasture-fed lambs in both New Zealand (Sutherland et al. 1979) and the
United Kingdom (Sargison et al. 2001). Its aetiology is unclear and may reflect inhibition, by
increasing concentrations of methylmalonyl Co-A in mitochondria in the liver, of β-oxidation of
fatty acids (Brindle et al. 1985) but also an inability to produce very low-density lipoproteins for
export of lipid from the liver (Kennedy et al.1997). Phosphatidyl choline is important for the
synthesis of very low-density lipoproteins (Yao and Vance 1988), demand for which is likely to be
increased as the anorectic Co-deficient lamb mobilises body lipid. Given the scarcity of choline
from dietary sources, the need for an increase in the step-wise methylation of phosphatidyl
ethanolamine to produce phosphatidyl choline could be anticipated.

The possibility exists that in pasture-fed lambs, utilisation of Hcy in other pathways prevents its
accumulation despite a deficiency of methylation. Hcy also serves as an intermediary in the
conversion of methionine to cysteine via the trans-sulphuration pathway. It has now been
established that the gastrointestinal tract is a significant ‘first-pass’ site of metabolism of
methionine in sheep (Lobley et al. 2003), and a significant site of synthesis and trans-sulphuration
of Hcy in young pigs (Reidijk et al. 2007). High rates of trans-sulphuration would be consistent
with a role for cysteine in fuelling the significant immune capability of the gastrointestinal tract.
Cysteine is important as a component of mucins secreted by goblet cells and for the provision of
glutathione in the management of oxidant stress of proliferating epithelial and immune cells.
Grazing animals will inevitably have greater exposure to bacterial, protozoal and helminth
pathogens on pasture than would be expected in pen-fed sheep. It seems plausible that the demands
of the immune response in the alimentary tract for utilisation of methionine and Hcy via the trans-
sulphuration pathway will compete with re-methylation reactions to limit accumulation of Hcy. If
this is the case, it might explain both the low concentrations of Hcy in plasma observed in the free-
grazing ewes and lambs in the current study, and the relatively small response of Hcy compared
with MMA in the pasture-fed sheep in the study by Vellema et al. (1999), compared with responses
in pen-fed sheep and cattle. It may also explain some of the difficulties long-acknowledged in
predicting response to supplementation from low concentrations of vitamin B12 in plasma of grazing
sheep (Clark et al. 1989). Kinetic studies of S-amino acid metabolism in grazing ruminants would
be needed to verify this hypothesis.

In conclusion, lack of change in concentrations of Hcy in plasma of ewes and lambs during a
responsive vitamin B12 deficiency suggests that either the homocysteine-methionine methylation
pathway is much more highly conserved than the propionate-succinate pathway in vitamin B12
deficiency, or the sensitivity of Hcy as an indication of methylation deficiency can be masked by
demand for Hcy for the trans-sulphuration pathway. The latter hypothesis is worthy of further
exploration. At this stage, however, measurement of concentrations of Hcy in plasma does not seem
to offer the possibility of improved prediction of response to supplementation in Co-vitamin B12
deficient lambs on pasture.

Acknowledgement

The authors thank Meat & Wool New Zealand for funding these studies.

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1988

Submitted 07 July 2009

Accepted for publication 05 November 2009

Figure 1. Schematic illustration of methionine metabolic pathways via trans-methylation
(TM), from S-adenosyl methionine (SAM) to ??? (SAH), trans-sulphuration (TS), re-
methylation (RM), protein synthesis (S) and breakdown (B), and the role of methyl-
cobalamin (MC) and betaine-homocysteine methyltransferase (BHMT) in methyl-group
transfer from methyl-tetrahydrofolate and betaine (MTHF). Adapted from Shoveller et al.
(2005).

Figure 2. Changes in the mean (a) liveweight (kg), and concentrations of (b) vitamin B12
(pmol/L), (c) methylmalonic acid (MMA; µmol/L), and (d) total homocysteine (mol/L) in the
plasma of supplemented lambs suckling supplemented ewes (□), unsupplemented lambs
suckling supplemented ewes (■), supplemented lambs suckling unsupplemented ewes (◊),
and unsupplemented lambs suckling unsupplemented ewes (♦). Vertical bars represent the
SE for the respective overall means.
 Proteins
S B

MTHF Methionine
SAM Polyamines
MC
RM TM CH3
CH3
BHMT SAH

Homocysteine
Betaine
TS
Serine
Choline
Cystathionine
Sulphate
α-Ketobutyrate
Taurine
Cysteine Glutathione
 a

45

40

35

Liveweight (kg)
30

25

20

15

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0
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Days

b
V
3
5
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ia
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c
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(
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 a

45

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0 50 100 1 50 200

3000

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c
80

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d
 5

4.5

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