THE LANCET
`
`Wheatley SA, Miller JM, jndad AR. Reducfion of post-operative pain
`jet ieparoscopic orbs! steriiiv-stop with local l.'||.'piVfl¢‘%!!’!¢'
`:1
`randomized, panllel, double-blind trial. Br] Obxrar G_ynaa:ol 19945
`101: -143-46.
`Mcken:n'e R, Phitayakorti P, Uy NTL, Wadhwa RK, Vicmie AF.
`Topical etidocaine during laparoscopic tubal occlusion for post-
`operative pain relief. Obrur Gynacol 1986; 67: 447-49.
`Cook Fl‘, Iambert '11’. An investigation of the eifectiveness of
`bupivacaine applied to the endomen-ial wall and fallopian tubes in
`reducing pain after laparoscopic sterilization. Anenh Inmu Care 1986;
`14: 148-51.
`Smith BE, MacPherson GH, De Jonge M, Grifliths IM. Rectus sheath
`and mesosalpinx block for laparoseopic sterilization. Anus-zheria I991;
`46: 875-77.
`10 Helvaeioglu A, Wei; R. Operative laparoaoopy and post-operative pain
`relief. Rm? Sun? 1992; 57: 549-52.
`CD, '\'v“ethcheier B'v', Thompson RE. Bupivacaine infiitration
`of the mesosalpinx in ambulatory surgical laparoscopic tubal
`srerilimtion. Can ]/lnaasth 1987; 34: 362-65.
`12 Narch P, Benhatnou D, Fernandez H. lntra-peritoneal local anaesthetic
`fior shoulder pain afier day-care laparoscopy. Lancet 1991; 338:
`l 569-70.
`13 Benhamnu D, Nlrchi P, Mamit IX, Fernandez H. Post-operative pain
`
`ll
`
`alter local anesthetics for lapamseopic sterilization. Obsur Gynecol
`1994; 224: 377.30‘
`14 Edwards ND. Barclay K, Carling S], Martin DG, Morgan MA. Day
`care laparoscopy: I survey of post-operative pain and an assessment of
`the value of diciofenac. Anaesthesia 1991; 46: 1077» 80.
`15 Levenson C], Daily H1, Marko MW, Richardson DC. Noneleetric
`loparoscopic sterilization: experience with a siiasuc band. Obmt
`Gynawl I976; 48: 494—96.
`15 Filshie C-M, Casey D, Pcgrsore IR. % t:'tani-.:.'ri!:'.i!ie:rn robber all
`female sterilization Br] Obszzr Gynaecol 1981; 88: 655-62.
`17 Millar ]M. Fitness for discharge after day surgery. Anasrlmia 1988; 43:
`4 l B.
`18 Collins KM, Doeherry PW, Planter-in OM. Post—operative morbidity
`following gynaecological out—patient lapnroseopy: I reappraisal of the
`service. Anaarheno 1984; 39: 819-22.
`:9 Spic' an F, Hulk IF, Ost.‘.cirnc.- CW, Mueller RA. Pi-.armacn‘.:-1.-.'.‘tica
`and pharmacodynamies of local analgesia for lapuroscopic tubal
`ligations. Am J Obsru Gynawl I983; 146: 821-24.
`20 Pclland PC. The application of lidocnine to the fallopian tube during
`tubal fulguration by lapamscopy. Obsm Gynacul 1976; 47: 501-02.
`21 Deeb R], Viechnicki B. Laparoscopic tubal ligation under peritoneal
`lavage lnaestheisa. Reg/lnasrrlr I985; 10: 24-27.
`
`for
`
`Effects of vitamin B12, folate, and vitamin B6 supplements in
`
`elderly people with normal serum vitamin concentrations
`
`Hans J Naurath, Etienne Joosten, Reiner Riezler, Sally P Stabler, Robert H Allen, John Lindenbaum
`
`Suunay
`in a prospective. multicentre, doubleblind controlled study,
`the effect
`of an intramuscular vitamin
`supplement
`containing 1 mg vitamin 812, L1 mg folate, and 5 mg
`vitamin B6 on serum concentrations of methylmalonic acid
`(MMA), homocysteine (HCYS), 2-methylcitric
`acid (2-
`MCA). and cystathionine (CYSTA) was compared with that
`of placebo in 175 elderly subjects living at home and 110
`in hospital. Vitamin
`supplement
`and
`placebo were
`administered eight times over a 3week period. Vitamin
`supplement but not placebo significantly reduced all four
`metabolite concentrations at the end of the study in both
`study groups. The maximum effects of treatment were
`usually seen within 5-12 days. Initially elevated metabolite
`concentrations returned to normal in a higher proportion of
`the vitamin than of the placebo group: 92% vs 20% for
`HYCS; 82% vs 20% for MMA: 62% vs 25% for 2-MCA; and
`42% vs 25% for CYSTA.
`The response rate to vitamin supplements supports the
`notion that metabolic evidence of vitamin deficiency Is
`common in the elderly. even in the presence of normal
`serum vltamln
`levels. Metabolite
`assays
`permit
`identification of elderly subjects who may benefit from
`vitamin supplements.
`Lancet 1995; 346: 85-89
`
`Dopartrnent of Geriatric Medicine, University Wilton-Haddocks,
`Velhort. Gorrnmy (H J Naurath Mo); Department of Internal
`Motliclno, Division oi Geriatric Medicine, University Hospital KU
`Leuvon, Leuven. Iielglum (Pror E Joosten Mo): seven-Mod anion.
`Monster. Germany (R Rlezler Mo); University of Colorado Health
`science center, Division of Hematology, Denver (Prof S stabler Mo,
`Prof R H Allen Mo): lid Department oi Modlolne. Columbia
`University, college of Physicians and Surgeons, New York. USA
`(Profl Lindenbaum MD).
`correspondence to: Prof E Jousten. University Hospital KU Leuven.
`Brusselsestraat 69. 3000 Leuven. Belgium
`
`Vol 346 °]uly 8, 1995
`
`Introduction
`Methylrnalonic acid (MMA), homocysteine (HCYS),
`cystathionine (CYSTA), and 2-methylcitric acid (2-
`MCA) are metabolites in enzymatic reactions dependent
`on vitamin B12, folate, and vitamin B6.” Vitamin B12
`(as 5'-deoxyadenosylcobalamin) is an essential cofactor in
`the enzymatic conversion of methylmalonyl-CoA to
`succinyl-CoA.’ The conversion of methylmalonyl-CoA to
`MMA results in an elevated serum MMA concentration
`in
`vitamin B12
`deficiency.
`Folate
`(as methyl-
`tetrahydrofolate) is required for methylation of HCYS to
`methionine
`catalysed by methionine
`synthase with
`vitamin B12 (merhylcobalamin) as a cofactor. Vitamin B6
`(as cofactor pyridoxal-5'-phosphate) is required for trans-
`sulphuration of HCYS through cystathioninc synthesis, a
`HYCS catabolic pathway.‘ 2-MCA concentrations are
`raised in the serum of most patients with vitamin B12
`deficiency. Concentrations of CYSTA are elevated in
`most patients with deficiencies of vitamin B12 and folate,
`and may be increased in B6 deficiency.“
`In an epidemiological study,’ we demonstrated that the
`prevalence of elevated concentrations of these metabolites
`in an elderly population was higher than the prevalence of
`low serum vitamin B12, folate, and vitamin B6 values. We
`postulated that tissue deficiency of these vitamins (shown
`by elevated metabolite concentrations) may be more
`common than that estimated by measuring serum vitamin
`levels.
`Intracellular
`vitamin
`deficiency might
`be
`subclinical, but even in patients with normal serum
`vitamin values, clinical and haematological
`signs of
`vitamin
`deficiency may
`be
`found with
`elevated
`concentrations of metabolites.” An elevated HCYS
`concentration
`is
`an
`independent
`risk
`factor
`for
`atherosclerotic diseases."*" The prevalence of elevated
`metabolite concentrations and potential risk factors for
`related
`disease,
`such
`as
`neuropsychiarric
`and
`thromboembolic disorders, is also high in the elderly.
`
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`
`living at been
`Vitamin
`Placebo
`group
`group
`l"=38)
`(n=87)
`20 (23)
`22 (25)
`42 (48)
`43(49)
`16 (18)
`23 (26)
`13 (15)
`12 (14)
`
`54 (61)
`17 (19)
`
`52 (59)
`13 ( 15)
`
`In tiuepltd
`Vitamin
`group
`("=55l
`21(38)
`32 (58)
`16 (29)
`5 (9)
`
`19 (35)
`18 (33)
`
`Diabetes mellitus
`lschaemic heart disease
`Chronic heart failure
`Chrome obstructive
`lung disease
`Hypertension
`Neuropsycmatrlc
`disorders
`
`Number ()6) of subjects: some had more than one dimnosls.
`Table 1: Dlaptoeee In elderly euojecta
`
`We assessed whether vitamin supplements would lower
`serum concentrations of I-ICYS, CYSTA, MMA, and 2-
`MCA in an elderly population by a double-blind placebo-
`controlled study with a parenteral vitamin combination.
`
`Subjects and methods
`Study population
`During an 8-month period, 300 parLicipauLs aged 65-96 years
`were enrolled at 13 centres. None had participated in the
`previous epidemiological study.’ 180 were living at home and 120
`in nursing homes or acute geriatric hospitals. Subjects living at
`home were recruited by telephone from a general pracutionefs
`register. They were able to carry out all normal daily activities
`independently or with minimal help and were not acutely ill. A
`history of concomitant diseases was assessed from the medical
`records supplied by their general practitioner. Patients living in a
`nursing home or admitted to hospital were randomly chosen to
`participate in the study. We excluded patients who had taken
`vitamins during the previous 3 months or who had a life-
`threatening disease, history of a
`coagulation disorder or
`treatment with anticoagulants, hypersensitivity to lignocaine, or
`severe renal insuficiency (creatinine clearance of less than 30
`mlJmin").
`The participants received intramuscular injections of 1 mg
`hydroxocobalamin,
`l-1 mg folate,
`and 5 mg pyridoxine
`hydrochloride in 4 mL water (vitamin group), or the placebo
`preparation containing 0-4 mg azorubin, 36 mg sodium chloride,
`and 4 ml. water (placebo group). The two preparations were
`similar in appearance and were supplied by Medice, Chem
`Pharmazeutische Fabrik (Iserlohn, Germany). Before injection,
`both the test drug and the placebo were mixed with 24 mg
`lignocaine hydrochloride in 1 mL water. Each participant
`received an intramuscular injection in the morning of days 1, 3,
`5, 8, 10, 12, 16, and 20. Fasting blood samples were taken before
`the first injection on day 1 and before injections on days 5, 12,
`and 21. The study design was double-blind. All patients were
`included in the analyses if they received at
`least the firct 5
`intramuscular injections, with follow-up analysis after 3 weeks.
`The protocol was
`approved by the Freiburger Ethik
`Kommission in Germany and the ethics committees of the
`participating academic centres. lnforrned consent was obtained
`from all subjects before they joined the study. All patients
`underwent a complete history and physical examination.
`
`Laboratory investigations
`Venous biooci was obtained in the morning after an overnign'' t
`fast. Laboratory tests were done in the same laboratories as
`described previously.’ Serum HCYS, CYSTA, MMA, and
`2-MCA were assayed by capillary gas chromatography and mass
`spectrometry. Serum vitamin B12 and folate were measured by a
`quantitative radioassay method with a purified intrinsic factor
`and a purified folate-binding protein. Vitamin B6 was measured
`in deproteinisecl serum by a radioenzyrnatic method with tyrosine
`spodecarboxylase and “C-tyrosine. The nonnal reference ranges
`for the serum vitamins and metabolites were calculated as within
`2 SD of the mean in 99 healthy volunteers aged 20-25 years’:
`vitamin B12 103-406 prnol/L; folate 5-4-16-3 nmol/L; vitamin
`B6 28-7-l 62 nmol/L; HCYS 5-0-13-9 pmol/L; CYSTA 72-245
`nmol/L; M.MA 62-247 nmol/1.‘ 2-MCA 62-192 nmol/L.
`
`Analysis
`Baseline values and the response to vitamin supplemts were
`examined separately in the subjects at home and in hospital. Data
`were log transformed before analysis. Means were compared by
`Student’: 1 test for the evaluation of the baseline characteristics
`and ANOVA with repeated measures correcting for the baseline
`value for the changes in the metabolites after therapy. A multiple
`linear regression model was used to assess the influence of
`vitamin
`supplements
`versus
`placebo
`on
`the metabolite
`concentrations between days I and 21. The x’ test was used to
`compare frequency
`‘attributions bet-weer: groups. A p value at
`0-05 was considered statistically significant.
`
`Results
`Study groups
`Of the 300 participants, 180 were living at home and 120
`were in hospital or nursing home. They were randomly
`assigned to vitamin supplement or placebo so that there
`were equal numbers in the two treatment arms.
`15
`subjects were
`excluded from analysis because
`of
`incomplete follow—up or missing results: ten subjects in
`hospital (5 in each group) and five living at home (1
`vitamin and 4 placebo). 285 participants completed the
`trial (table 1). Vitamin and metabolite concentrations
`before treatment did not differ between the vitamin and
`placebo groups (table 2), except that among subjects in
`hospital CYSTA concentrations were higher
`in the
`placebo group (p=O-05). The prevalence of elevated
`serum metabolite
`and
`subnormal
`serum vitamin
`concentrations was generally well balanced between the
`groups (table 3), ald-tough there were more subjects with
`elevated HCYS levels in the placebo group than in the
`vitamin group among those living at home (p=0-006).
`
`Effect of supplements on metabolite concentrations
`The figure shows the effect of vitamin and placebo
`injections
`on
`serum concentrations
`of
`the
`four
`metabolites. In the analysis, these values were corrected
`for the corresponding baseline value (metabolite level on
`
`characteristic
`
`Mde/hind-
`
`|.Mn¢ethome
`Vitamin group
`
`age h {ail "
`Mean
`Geometric mean (95% range) serum concentrations
`Vitanin 812 (pmol/L)
`199 (38450)
`10-9 (3-4-36)
`Folate (nrnoI/ L)
`Vliunln 86 (nmol/L)
`65 (19-221)
`11-6 (7-19-2)
`HCY5 (jl.mol/L)
`222 (55-751)
`MMA (nmol/L)
`252 (102-625)
`CYSTA (r\mol/L)
`168 (78—362)
`2 MCA (nmol/L)
`Table 2: characteristics In the study groups before treatment
`
`Placebo group
`41/46
`
`"1571-(6-1)
`
`189 l 76-476)
`95 (34-26)
`63 (13-227)
`12-6 (8-1-26)
`243 (84-697)
`256 (92—712)
`16? (70-399)
`
`In hospital
`Vntanln group
`9/46
`79-3 (8-7)
`
`207 (66-647)
`34 (25-28)
`42 (13-142)
`13-2 (7-1-25)
`227 (68-762)
`280 (111-710)
`165 (8C.L3-10)
`
`Placebo group
`19/36
`80-4 (8-5)
`
`207 (86-495)
`8-4 (2-5-28)
`42 (10-167)
`13-7 (74-254)
`254 (57-1129)
`346 (99-213)
`193 (50-471)
`
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`
`flflnflhlb
`
`Toiii (I'I=175)
`12 (7)
`22 (13)
`17 I 10)
`77 (44)
`50 (29)
`83 (50)
`30 (34)
`
`Uvlflflhollli
`Placebo group (r‘t=87)
`Viianin group (n=88)
`7 (8)
`5(6)
`Vltunln B12 <103 pmol/L
`10 (11)
`12 (14)
`Falate '54 nmol/L
`10 (11)
`7(8)
`Vnarntn B6 <28-7 nmol/L
`44 (50)
`33 (37)
`MMA I247 Ilml/L
`33 (38)’
`17(19)
`HCYS >13-9 pmol/I.
`42 (48)
`46 (52)
`CYSIA 3245 nmul/L
`29 (3:3)
`31' (35)
`2-MCA 3192 nmol/L
`Nurvl1er(%) of subjects. ‘p=0-006 for comparison with vitamin group.
`Table 3: Abnormal serum concentrations of vltemlne and metabolites belore treatment
`
`II lI%hl
`Vliull
`gruiiir (I‘t=55)
`5 (11)
`12 (22)
`10 (1.8)
`17 (31)
`?7 (40)
`
`Flaaabo group it-=55)
`4 (7)
`9 (16)
`14 (25)
`27 (50)
`27 (50)
`41 (75)
`73 (47)
`
`v.._- ._ .am
`lutul [Il=J.J.U|
`
`10(9)
`21 (19)
`24 (22)
`44 (40)
`49 (45)
`74 (57)
`43 (44)
`
`In beepltel
`‘Jitanin amen
`
`Plaza‘-.3 group
`
`14/71 (20)
`41/50 (82)
`12/60 (20)
`36/39 (92)
`21/83 (25)
`33/79 (42)
`14/55 (25)
`33/53 (62)
`Table 4: Frequency dletrlbutlon ol patlente with elevated pretreatment metabolite values but normal values on day 21
`
`20/22
`12/33
`13/22
`
`day 1). The difierence in concentrafions between the
`vitamin supplement and placebo groups was greatest at
`day l2 for all metabolites in subjects living at home and in
`hospital.
`To investigate the effect of vitamin supplements on
`metabolite
`concentrations,
`pretreatment metabolite
`concentration, age, sex, and group were brought into a
`multiple linear regression model. Among subiects living at
`home and in hospital, there was a significant effect of the
`vitamin treatment over placebo in the concentrations for
`each of the fear metabolites on day 21. Age was
`significant only for MA. The numbers and percentages
`15
`
`elevated metabolite
`initially
`with
`patients
`of
`concentrations that became normal after vitamins or
`placebo are shown in table 4. For MMA, HCYS, and 2-
`MCA the concentrations reached the normal range in
`significantly greater proportions of
`those
`receiving
`vitamins than of the placebo group. More than 80% of
`the elevated MMA and HCYS concentrations were
`normal at the end of the study after vitamin supplements;
`2-MCA concentrations became normal in more than half
`and CYSTA in 42%. In vitamin-Lreal.ed patients whose
`metabolite concentrations had returned to normal at day
`21, a normal concentration was achieved by day 5 (after
`
`§ §
`
`
`
`Semm2-MCA(nmol/L)SerumCYSTA(nmol/L) w8
`
`
`
`
`
`SerumMMA(nrwol/L)SerumHCYS(pmollL)
`
`Day cl study
`
`1
`
`Day of study
`
`Figure: Serum metabolite eoneerrtntlona after vltamln supplementation (- - -) or placebo (—) In elderly subjects (a) at borne and (b)
`in ltoepital
`values represent geometric means. error bar antilog of (mean. SEM) logarithmically transformed data
`
`Vol 346 ‘July 8,1995
`
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`
`THE IANCET
`
`only two vitamin injections) for MMA, HCYS, CYSTA,
`and 2-MCA, respectively, in 72% (19/26), 88% (14/l6),
`72% (15/21), and 62% (13/21) of the subjects living at
`home and 100% (15/15), 65% (13/20), 33% (4/12), and
`46% (6/13) of those in hospital.
`
`Effects of vitamin supplements In patients with normal
`serum vitamin concentrations
`
`Of the three vitamins studied, only deficiency of vitamin
`Bl2 would be expected to cause an elevated MMA. More
`than 80% of the subjects with initially high MMA had
`normal
`serum vitamin B12 concentrations
`at
`the
`beginning of the study (at home, 29/33 in the vitamin
`supplement group and 39/44 in the placebo group; in
`hospital, 13/17 in the vitamin supplement group and
`24/27 in the placebo group). The MMA fell to normal in
`33 (79%) of the 42 vitamin-treated subjects (home 22/29;
`hospital ll/13) compared with l2 (19%) of 63 placebo
`recipients (home 7/'39; hospital 5/24) (p<0-0001),
`Deficiency of any of the vitamins included in the
`supplement might
`have
`led
`to
`raised HCYS
`concentration. Of the subjects with increased HCYS, 19
`had normal pretreatment serum values of all
`three
`vitamins. HCYS concentrations became normal after
`vitamin supplements in 16 (home 8/9; hospital 3/10)
`compared with only 7 of 35 placebo recipients (home
`4/20; hospital 3/1 S) (p<0-0001).
`
`Discussion
`intramuscular supplements with
`Our data show that
`vitamin B12, folate, and vitamin B6 reduced raised serum
`levels of HCYS and MMA in the majority of elderly
`subjects who had no overt
`chronic
`renal
`failure,
`irrespective of the serum vitamin concentrations. Serum
`2-MCA also fell
`to within the normal range in most
`subjects, and CYSTA concentrations fell to normal in
`nearly half. Similar responses were noted in the subjects
`living at home and in hospital.
`The proportion of subjects with initially elevated
`metabolites whose values fell to normal by the end of the
`study was significantly higher in the vitamin group than
`the placebo group for all four metabolites. Other studies
`have established the value of measuring serum MMA and
`HCYS in the diagnosis of clinically significant vitamin
`B12 and folare deficiency and in follow-up after relevant
`vitamin substitution.‘-’“-"'” Furthermore, many studies
`have shown that elevated homocysteine is an independent
`risk factor for cerebral, coronary, and peripheral vascular
`disease."’'" In addition to vitamin B12 and folate, vitamin
`B6 plays an important role in the degradation of HCYS
`since it is a cofactor for cystathionine-B-synthase,
`the
`enzyme that converts HCYS to cystathionine. Serum
`concentrations of HCYS and CYSTA are elevated in rats
`deficient
`in
`vitamin B6,"
`and
`serum CYSTA
`concentrations are increased it: the majority of patients
`with vitamin B12 and folate deficiency,’ as are 2-MCA
`values in those with vitamin B12 deficiency.‘
`Our schedule and dosage of 1 mg vitamin B12 and
`1-1 mg folate administered in eight
`injections over 3
`weeks is suflicient to treat deficiencies of these vitamins.
`However, for vitamin B6, the 5 mg dose used is lower
`than that of previous studies of oral B6 treatment,”-"
`although the recommended dietary allowance is 2-0 mg
`for men and 1'6 mg for women over age 51 .’° It is possible
`that a higher dose of parenteral vitamin B6 would have
`had a greater effect on the HCYS and CYSTA levels in
`
`our subjects. However, serum B6 levels rose to above the
`normal range (ie, >162 nrnol/L) by day 21 in ii subjects
`who had had raised pretreatment values; nonetheless,
`serum CYSTA concentrations remained elevated in 5 of
`the 11 patients.
`We’ and others“ have found that the majority of the
`healthy elderly subjects with elevated MMA levels have
`normal vitamin B12 levels. Moreover, in 79% of patients
`with a normal serum vitamin B12 concentration and an
`elevated MMA level in the present study, the MMA fell to
`normal within 3 weeks of vitamin supplements,
`in
`contrast to only 19% of the patients treated with placebo.
`The results for patients with elevated HCYS and normal
`levels of all three vitamins were equally striking. Hence,
`our findings add further support to the view that many
`elderly people have metabolic evidence of early vitamin
`B12, folate, or vitamin B6 deficiency despite normal
`serum vitamin
`concentrations.
`Serum metabolite
`concentrations were also found to be a more sensitive
`indicator of early vitamin B12 deficiency than vitamin
`levels in patients with pernicious anaemia who were
`withdrawn from monthly maintenance therapy.‘
`In
`addition,
`about
`5% of patients with unequivocal
`haematological or neurological syndromes caused by
`cobalamin deficiency have normal serum vitamin B12
`values at the time of presentation although serum MMA
`and HCYS are increased.‘
`fell
`Elevated MMA and HCYS concentrations
`substantially within 5 days after only two injections, with
`a maximum efiect seen by day 12. The clinical use of this
`response during substitution might be a rapid and
`objective
`test
`to confirm the presence of vitamin
`deficiency. Although decreases in concentrations of MMA
`and 2-MCA were presumably caused by vitamin B12, it is
`not possible from our study design to indicate which
`particular vitamin brought about the reductions in HCYS
`and CYSTA concentrations.
`Other factors may alter serum concentrations of the
`four metabolites. Although patients with frank renal
`failure were excluded from our study, mild to moderate
`renal dysfunction may have contributed to elevations in
`metabolites in some subjects,"'“ especially for CYSTA
`and 2-MCA,“ in which the responses
`to vitamin
`supplements were less uniform.
`Increases in HCYS
`concentrations may also be seen after treatment with
`various drugs,""" and any of the metabolites may be
`affected by inborn errors of metabolism.“-‘3-"’
`The response to vitamin supplements in patients with
`elevated MMA and HCYS and normal serum vitamin
`B12,
`folate, and vitamin B6 values, provides further
`evidence that vitamin deficiency in the presence of normal
`serum levels is more common than previously thought. To
`what extent reduction of these metabolite concentrations
`to normal values is associated with clinical improvement
`remains unknown.
`We thank Ms A L Nutting (Tulsa, Oklahoma, USA), M: Pvan Bergen
`(Munster, Germany), and Dr A van den Berg (Velp, the Netherlands) for
`their assistance. The statistical analysis was performed by ProfE Lesafie,
`Department of Epidemiology, Biosratisrical Center, KU Leuven
`(Belgium). This study was supported by a grant from Medice.
`Chem Pharmazeurlsche Fubrik, lserlohn. Germany, but the company was
`not involved in the acquisition or management of the data.
`
`Roioroncu
`I Stabler SP, Marcel] PD, Podell ER, Allen RH, Lindenbcum I. Assay of
`rnethylmalonic acid in the serum of patients with cobalamin deficiency
`using capillary gas chromatography-mass spectromeuy. j’Cix'n Invest
`1986; 7: 1606-12.
`
`88
`
`Vol 346 - July 8. 1995
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`
`Slabler SP, Marcell PD, Podell ER, Allen RH, Savage DG,
`Lindenbaum J. Elevation oftotal homocysreine in the serum ofpatients
`with cobalamin or folate deficiency detected by capillary gas
`chromatography-mass specn-ometry. J Clin Invest 1988; 81: 466-74.
`Stabler SP, lindenbaum I. Savage DG. Allen RH. Elevation of serum
`eysuttltionine levels in patients with cobalaunin and folatc deficiency.
`Blood I993; 81: 3404-13.
`Allen RH, Stabler SP, Savage DG, Lirtdenbaum J. Elevation of
`2-ntethylcitric acid I and II in the serum, urine and cerebrospinal fluid
`of patients with cobaiamin deficiency. ivletaboiisnt i993; 41: 9'78 38.
`Rosenberg ll-l, Fenton WA. Disorders of propionate and
`methylmalonate metabolism. In: Scriver C, Beaudet: AL, Sly V/L,
`Valle D, eds. Metabolic basis of inherited diseases. New York:
`McGt-aw-Hill, 1989: 821-44.
`Mudd SH, Levy I-IL, Sltovby F. Disorder of uanssulfuration. 1n:
`Scriver C, Beaudet AL, Sly WL, Valle D, eds. Metabolic basis of
`New ‘.’ar‘.:: .‘v'.cGr:'w'-Hill, BB9: 693-734.
`Joosren F, vd Berg A, Rialer R, et al. Metabolic evidmce that
`deficiencies of vitnmin B12 (cobalntnin), folate and vitnmin B6 occur
`commonly in elderly people. Am] Clin Nurr 1993; 58: 468-76.
`Liodenbaurn J, Savage DG, Stabler SP, Allen R!-I. Diagnosis of
`cobalamin deficiency ll: relative sensitivities of serum cobslarnin,
`methylmalonic acid. and total homocysteine concentrations.
`Am,7 Herrera! !9905 34: 99-107.
`Savage DG, Lindenbaum I, Stabler SP, Allen RH. Sensitivity of serum
`tuedtylmalouic acid and total homocysteine determinations for
`diagnosing cobalamin and folate deficiencies. Am ] M41994; 96:
`239-46.
`I0 Clarke R, Daly L. Robinson K, et al. Hyperhornocysteinernia: an
`independent rislt factor fun‘ vascular disease. N Eugi} Med 1991; 324:
`1 149-54.
`ll StampferMJ, Malinow MR, Willett WC, et al. A prospective study of
`plasma hornocyst(e)ine and risk of myocardial infarction in US
`physicians. JAMA l992; 268: 877-81.
`I2 Brartstrom L, lsraelsson B, Norrving B, et al. Impaired homocysteine
`metabolism in early-onset cerebral and peripheral occlusive arterial
`disease. Efiecrs of pyridoxine and folic acid treatment. Atherosclerosis
`1990; 81: 5l-60.
`l3 Ueland PM, Reftum H. Plasma ltomocysteine, a risk factor for vascular
`disease: plasma levels in health, disease and drug therapy.
`Jbala Clirt Medl989; 114:-173-501.
`l4 Ueland PM, Refsum H, Brattsn-om L Plasma homocysteine and
`cardiovascular disease. In: Francis RB, ed. Atherosclerotic
`
`Hormone replacement therapy and serum
`angiotensin-converting-enzyme activity in
`postmenopausal women
`
`Anthony J Proudler, A I Hasib Ahmed, David Crook,
`lgnac Fogelman, Janice M Rymer. John G Stevenson
`
`The mechanisms by which hormone replacement therapy
`(HRT) reduces the risk of coronary heart disease (CHD) are
`incompletely understood, but may include direct arterial
`effects. We examined the effect of oestrogen/progestagen
`HRT
`on
`serum angiotensin-convertingenzyrne
`(ACE)
`activity in postmenopausal women. After 6 months. ACE
`activity was reduced by 20% (p<0-001) on average in 28
`treated women but remained unchanged in 16 controls.
`Serum ACE activity is modifiable by gonadal steroids and
`changes in serum ACE may represent a novel mechanism
`by which HRl reduces CHD rlsk in women.
`Lancet 1995: 348: 89-90
`
`Oestrogen replacement therapy is associated with reduced
`risk of coronary heart disease (CHD) in postmenopausal
`women.‘ Whilst
`beneficial
`changes
`irt
`lipid
`and
`carbohydrate metabolism, body-fat distribution,
`and
`
`Vol 346 - July 8, 1995
`
`THE LANCET
`
`cardiovascular disease, hemostasis and endothelial function. New York:
`Marcel Delrker, 1992: 183-236.
`15 Boers GH], Smala AGH, Triibels FJM. Heterozygoeiry for
`homocystinuria in premature peripheral and cerebral occlusive arterial
`disease. N Engl_7Med I985: 313: 709-15.
`I6 Cocltroft DW, Gault Ml-I. Prediction of creatinine clearance from
`serum creatinine. Nephnm 1976; 16: 31-41.
`l7 Ueland PM, Refsum H, Stabler SP, Malinow MR, Andersson A,
`Allen RH. Total homocysteine in plasma or serum: methods and
`clinical applications. Clin Chan I993; 39: 1764-79.
`I8 Lindenbaum J, Healton EB, Savage DG, et al. Neuropsychianic
`disorders caused by cobalamin deficiency in the absence of anemia or
`macxocytosts. N Eng] Med 1988; 318: 1720-28.
`I9 Pennypaclter LC, Allen RH, Kr.:llyJP, ct al. Hifit prevalence of
`cobalnmin deficiency in elderly out-patients. _7Am Gsriotr Soc 1992;
`40: 1197-1204-.
`20 Joosten E, Pelernnns VI, Devos P, et al. Cobaltunin absorption and
`serum homocysteine and methylmalonic acid in elderly subjects with
`low serum cobalamin. Eur] Haematol 1993; 51: 2$30.
`21 Moelby L, Rasmussen K, Jensen MK, Pederson K0. The relationship
`between clinically confirmed cobalamin deficiency and suum
`mehylmalonic acid. 3 Inter Med 1990; 228: 373-78.
`22 Allen RH, Sutbler SP, Slvage DG, Lindenbaum J. Metabolic
`abnormalities in cobalnrnin (vitamin B»! 2) and folnte deficiency.
`FASEB I993; 72 I344 53.
`23 Brattstrom L, Israelsaon B, Lindgarde I, I-lultbcrg BL. Higher total
`plasma homccysteine in vitamin B-12 deficiency than in heterozygosity
`for homocystinnria due to cystathionine B-synthase deficiency.
`Mctabolimt 1988; 37: 175-78.
`24 Stabler SP, Allen RI-l. Elevations ofserum cystathionine and
`homocysteine in vitamin B6, folate and cobalamin deficient rats. Blood
`1994; 84: 1 lBa.
`25 Ubbink JB, Vermaak WJH, vd Mezwe A, Becher PJ. Vitamin B-12,
`vitamin B-6, and folate nutritional status in men with
`hyperhomocysteinemia. Am 3' Clin Nutr 1993; 57: 47-53.
`26 National Research Council. Recommended dietary allowance. 10th ed.
`Washington DC: National Academy Press, 1989.
`27 Llndenbaum J, Rosenberg IH, \Vilson PWF, Stlbler SP, Allen RH.
`Prevalence of cobalamin deficiency in the Framingham elderly
`population. Am] Ch‘): Nuzr 1994; 60: 2-1 1.
`28 Moelby L, Rasmussen K, Rasmussen HH. Serum methylmalonic acid
`in urunia. Stand] Clin Lab limes 1992; 52: 351-54.
`
`haemostasis may bring about this effect, there is also
`evidence for direct arterial efiects of oestrogen.‘ Elevated
`activity of serum angiotertsin-converting enzyme (ACE)
`and deletion polymorphism in the ACE gene may both be
`independently associated with increased risk of CHD in
`men." Serum ACE levels may vary four-fold between
`healthy individuals, with up to 50% of inter-individual
`variation
`attributable
`to
`the
`polymorphism.“
`In
`longitudinal studies, ACE concentration appears constant
`within groups composed predominantly of men.’ Whether
`serum ACE is similarly constant in women or may be
`influenced by menopause has not been studied. We
`hypothesised that circulating levels of gonadal steroids
`may determine ACE activity in women and that serum
`ACE activity may thus be a modifiable CHD risk-factor.
`Accordingly, we measured serum ACE activity in
`postmenopausal women before and during hormone
`replacement therapy (HRT) and in a control group of
`untreated women.
`The study population comprised the initial recruitment of
`eligible patients into a prospective HRT study. The population
`consisted of 28 postmenopausal women assigned to be treated
`with continuous combined HKl'
`(2 mg oestradiol valerate and
`0-7 mg norethistcrone orally daily) and 16 postmenopausal
`woman who did not wish to receive I-IKI‘, who were recruited
`concomitantly
`to
`serve
`as
`controls. All women were
`postmenopausal (>l2 months’ amenorrhoea), confirmed by
`elevated serum follicle-stimulating hormone, and none had
`received any oestrogen preparatioru within 12 months or
`implants within 3 years. The women were apparently healthy
`
`89
`
`Lilly Ex. 2069
`Sandoz v. Lilly IPR2016-00318