029
`
`Sensitivity of Serum Methylmalonic Acid and Total
`Homocysteine Determinations for Diagnosing Cobalamin
`and Folate Deficiencies
`DAVID G. SAVAGE, M.D., JOHN LINDENBAUM, M.D., New York, New York, SALLY P. STABLER, M.D., ROBERT H. ALLEN,
`M.D., Denver, Colorado
`PURPOSE: Patients with cobalamin (vitamin 812)
`deficiency usually lack many of the classic
`features of severe megaloblastic anemia;
`because of the low diagnostic specificity of
`decreased serum cobalamin levels, demon(cid:173)
`strating the deficiency unequivocally is often
`difficult. We examined the sensitivity of
`measuring serum concentrations of methyl(cid:173)
`malonic acid and total homocysteine for
`diagnosing patients with clear-cut cobalamin
`deficiency and compared the results with those of
`patients with clear-cut folate deficiency.
`PATIENTS AND METHODS: Serum metabolites were
`measured for all patients seen from 1982 to
`1989 at two university hospitals who met the
`criteria for cobalamin and folate deficiency states
`and for such patients seen from 1968 to 1981
`from whom sto.red sera were available. In all, 406
`patients had 434 episodes of cobalamin
`deficiency and 119 patients had 123 episodes of
`folate deficiency. Criteria for deficiency states
`included serum vitamin levels, hematologic and
`neurologic findings, and responses to therapy.
`Responses were documented in 97% of
`cobalamin-deficient patients and 76% of folate(cid:173)
`deficient patients. Metabolite levels were
`measured by modified techniques using capillary(cid:173)
`gas chromatography and mass spectrometry.
`RESULTS: Most of the cobalamin-deficient
`patients had underlying pernicious anemia; two
`thirds were blacks or Latinos. Hematocrits were
`normal in 28% and mean cell volumes in 17%. Of
`the 434 episodes of cobalamin deficiency, 98.4%
`of serum methylmalonic acid levels and 95.9% of
`serum homocysteine levels were elevated
`(greater than 3 standard deviations above the
`mean in normal subjects). Only one patient had
`normal levels of both metabolites. Serum
`
`From the Departments of Medicine, Columbia University, College of
`Physicians and Sugeons, Columbia-Presbyterian Medical Center, and
`Harlem Hospital Center, New York City (DGS, JLJ; Department of Medicine
`(SPS, RHAJ and Department of Biochemistry, Biophysics and Genetics
`(RHAJ, University of Colorado Health Sciences Center, Denver, Colorado.
`This work was supported in part by Department of Health and Human
`Services Research Grants (DK-21365 and AG-09834) from the National
`Institutes of Health.
`Requests for reprints should be addressed to Dr. John Lindenbaum,
`Department of Medicine, Columbia-Presbyterian Medical Center, 630
`West 168th Street, New York, New York 10032.
`Manuscript submitted December 29, 1992, and accepted in revised
`form April 8, 1993.
`
`March 1994 The American Journal of Medicine Volume 96
`
`239
`
`homocysteine levels were increased in 91 % of
`the 123 episodes of folate deficiency.
`Methylmalonic acid was elevated in 12.2% of the
`folate-deficient patients; in all but one, the
`elevation was attributable to renal insufficiency or
`hypovolemia.
`CONCLUSIONS: For the cobalamin-deficient
`patients, measuring serum metabolite
`concentrations proved to be a highly sensitive
`test of deficiency. We conclude that normal levels
`of both methylmalonic acid and total
`homocysteine rule out clinically significant
`cobalamin deficiency with virtual certainty.
`
`Cobalamin (vitamin B12
`
`) deficiency is conunon.
`Early diagnosis and treatment can prevent severe
`anemia or potentially irreversible damage to the ner(cid:173)
`vous system from developing, but the majority of
`patients lack the textbook features of advanced defi(cid:173)
`ciency of the vitamin [1--4). Serum cobalamin concen(cid:173)
`tration is widely used as a screening test for deficiency
`but has major limitations. Its sensitivity is less than per(cid:173)
`fect [5,6], and many workers [4,7-12) have found that
`a substantial proportion-perhaps 25% to 50% [4,12](cid:173)
`ofpatients with low serum cobalamin levels do not ap(cid:173)
`pear to be deficient in the vitamin or to have underly(cid:173)
`ing disorders predisposing them to deficiency.
`The metabolites methylmalonic acid and homocys(cid:173)
`teine accumulate when the two mammalian cobal(cid:173)
`amin-dependent enzymatic reactions are impaired
`[13,14]. We found that serum levels of both metabo(cid:173)
`lites are markedly elevated (more than three standard
`deviations [SD J above the mean in healthy control sub(cid:173)
`jects) in most deficient patients, suggesting that these
`tests are a useful and convenient way to establish a
`diagnosis of cobalamin deficiency [4,13-15]. The as(cid:173)
`says were modified in early 1988 to further increase
`their specificity and precision [16,17).
`We report here using the modified techniques to as(cid:173)
`say more than 400 patients with clinical evidence of
`cobalamin deficiency. To our knowledge, this series,
`accumulated over 21 years, is the largest group of pa(cid:173)
`tients with well-docwnented cobalamin deficiency re(cid:173)
`ported in the literature. For comparison, we assayed
`a series of 119 patients with folate deficiency. In pre(cid:173)
`vious studies involving relatively small numbers offo(cid:173)
`late-deficient patients, total homocysteine concentra(cid:173)
`tions were elevated in most cases [14,18,19].
`
`Teva – Fresenius
`Exhibit 1018-00001
`
`

`
`SERUM METABOLITES IN COBALAMIN AND FOLATE DEFICIENCY/ SAVAGE ET AL
`
`TABLE I
`Criteria for Cobalarnin Deficiency In 434 Episodes In 401 Patients
`Serum Cobalamin < 200 pK/ml. and
`Diagnostic Marrow
`Response
`to CblRx·
`+/or Blood Smear
`+
`+
`NA
`+
`NA
`+
`0
`+
`
`No. of Epi&Odes
`309
`13
`107
`5
`434
`
`%
`71.2
`3.0
`24.6
`1.2
`100
`
`Total
`
`TABLE Ill
`Criteria for Folate Deficiency in 123 Episodes in 119 Patients
`No. of Episodes
`123
`123
`123
`123
`94
`
`Diagnostic marrow or blood smear, or both
`Serum folate < 4 ng/ml •
`Serum cobalamin > 300 pg/ml t
`Underlying disorder associated with folate deficiency
`Documented response to folic acid
`
`~
`100
`100
`100
`100
`76.4
`
`•serum folate < 2.1 ng/ml in 99 episodes and 2.1 to 3.9 ng/ml in 24.
`tSerum cobalamin 301 to 350 pg/ml in 7 episodes, 351 to 400 pg/ml in
`10, 401 to 500 pg/ml in 20, 501 to 1,000 pg/ml in 60, and greater than
`1,000 pg/ml in 26. None of the patients had been treated wi1h vitamin 812
`before study.
`
`blood smears was defined as more than five cells with
`five lobes per 100 neutrophils or any cells with six or
`more lobes. Serum cobalamin levels were determined
`by radioassay using purified intrinsic factor (Quan(cid:173)
`taphase, Bio-Rad Laboratories, Richmond, CA) or by
`microbiologic assay with Lactoba.cillus leichmannii
`[27]. Serum folate concentrations were measured by
`milk-binder radioassays or by microbiologic as.say
`with Lactoba.cillus ca.sei [28].
`Serum concentrations of methylmalonic acid [29]
`and total homocysteine [30] were measured by mod(cid:173)
`ified [16,17] techniques using capillary-gas chro(cid:173)
`matography and mass spectrometzy. The nonnal
`ranges (calculated as the mean± 3 SD after log trans(cid:173)
`formation to correct for skewing toward higher val(cid:173)
`ues) are 53 to 376 nmol/L for methylmalonic acid and
`4.1 to 21.3 µmol/L for total homocysteine [16]. For 160
`of the patients with cobalamin deficiency and 19 with
`folate deficiency, serum metabolite levels were mea(cid:173)
`sured by methods used before our modifications were
`made, and the results were reported earlier [4,13-15].
`Standard statistical methods, including Student's
`t-test and the chi-squared test with Yates' correction
`for continuity [31), were used to analyze the data.
`RESULTS
`Criteria of Deficiency
`Serum methyhnalonic acid and total homocysteine
`levels were measured in 406 patients who had 434
`episodes of cobalamin deficiency; 25 patients had one
`or more recurrent episodes of deficiency because they
`discontinued maintenance treatment with the vitamin.
`The diagnostic criteria for including cobalarnin(cid:173)
`deficient patients in this series are swnmarized in
`Table I. All patients had low serum cobalamin lev(cid:173)
`els. Morphologic abnormalities (megaloblastic bone
`marrow or hypersegmented neutrophils, or both, with
`or without macroovalocytes, on blood smear) were
`found in 322 episodes; clear-cut responses to cobal(cid:173)
`amin therapy (as defined in Table II) were docu(cid:173)
`mented for 309 of the 322 episodes. Although marrow
`and blood films were not available for 107 episodes
`before therapy began, clear-cut responses were doc-
`
`NA = not available; Cbl = cobalamin; Rx = therapy.
`•See Table U for definition of responses.
`
`TABLE II
`Responses to Cobalamin Treatment In 434 Episodes of Cobalamin
`Deficiency in 409 Patients
`
`Decrease in mean ceN volume;;: 5 fl
`Increase in hematocrit ~ 0.05
`Resolution of thrombocytopenia
`Resolution of leukopenia
`Improvement in neuropsychiatric abnormalities•
`More than one of above responses to treatment
`Other responsest
`No documentation of response
`
`No. of Episodes
`369
`286
`110
`50
`112
`325
`9
`13
`
`~
`85.0
`65.9
`25.3
`11.5
`25.8
`74.9
`2.1
`3.0
`
`•Clear-cut improvement in neuropsychiatric abnormalities within the first
`three months of treatment. This was the only response to treatment in a
`single episode.
`tOnly one of the following responses was documented: reticulocytosis (five
`patients); correction of neutrophil hypersegmentation (three patients); and
`resolution of atrophic glossitis (one patient).
`
`PATIENTS AND METHODS
`Patients
`All cases of cobalamin and folate deficiency seen at
`Harlem Hospital Center and Columbia-Presbyterian
`Medical Center between July 1968 and June 1989 were
`reviewed. The majority of the patients were evaluated
`in consultation with one of the coauthors as part of
`prospective studies of megaloblastic anemias, and the
`hematologic and neurologic finding.s have been the
`subject of previous reports [4,13-15,20-26).
`Methylmalonic acid and total homocysteine levels
`were measured in all patients seen between 1968 and
`1981 who met the study's criteria for cobalamin and
`folate deficiency and from whom serum stored at
`-20°C was available. Fro.m 1982 to 1989, serum metab(cid:173)
`olite levels were measured in each of 267 consecu(cid:173)
`tively seen patients who met the cirteria for cobal(cid:173)
`amin deficiency (219 patients) or folate deficiency ( 48
`patients).
`
`Methods
`When available, smears of peripheral blood and
`bone marrow from cobalamin- and folate-de:ficient
`patients were coded and interspersed with those of
`patients without evidence of deficiency for blinded
`review by one of the authors. Hypersegmentation in
`
`240 March 1994 The American Journal of Medicine Volume 96
`
`Teva – Fresenius
`Exhibit 1018-00002
`
`

`
`SERUM METABOLITES IN COBALAMIN AND FOLATE DEFICIENCY I SAVAGE ET AL
`
`TABLEV
`Clinical Characteristics of 434 Episodes of Cobalamin Deficiency in
`406 Patients and 123 Episodes of Folate Deficiency in 119 Patients
`Cobalamin Deficienc~
`Folate Deficien9:
`No./fotaJt %
`No./fotalt %
`
`151/406
`255/406
`
`168/406
`135/406
`101/406
`2/406
`59/434
`313/434
`
`Characteristics·
`Sex
`Male
`Female
`Ethnicity
`Black
`White
`Latino
`Other
`Alcoholism
`Anemia
`Macrocytosis
`MCV> lOOfL
`334/402
`MCV > 110 fl
`224/402
`Leukopenia:t:
`73/433
`Thrombocytopenia :t
`136/384
`Pancytopenia
`48/384
`LDH > 1,000 U/l
`96/330
`Atrophic gfossitis
`111/434
`Neuropsychiatric syndrome§ 156/434
`
`37.2
`62.8
`
`41.3
`33.3
`24.9
`0.5
`13.6
`72.1
`
`83.1
`55.7
`16.9
`35.4
`12.5
`29.1
`25.6
`35.9
`
`70/119
`49/119
`
`58.8
`41.2
`
`105/119
`9/119
`5/119
`0/119
`107/123
`123/123
`
`68/91
`50/91
`22/123
`78/118
`21/118
`24/83
`27/123
`36/123
`
`88.2
`7.6
`4.2
`0.0
`87.0
`100.0
`
`74.7
`54.9
`17.9
`66.1
`17.8
`28.9
`22.0
`29.3
`
`TABLE IV
`Etiology of Cobalamin and Folate Deficiencies in 529 Patients
`Etiology
`Patients
`Cobalamin deficiency
`Pernicious anemia
`Proven
`Probable*
`Tropical sprue
`Proven
`Probablet
`Gastrectomy
`lleal disease or resection, or both
`Gastric and ileal resections
`Jejunal diverticula
`Vegetarianism
`Food cobalamin malabsorption
`Not established
`Total
`Folic acid deficiency
`Alcoholism
`Malnutrition
`Malabsorption syndrome
`? Oral contraceptives
`Pregnancy
`Sickle cell anemia
`Tropical sprue
`Total
`
`103
`11
`1
`1
`1
`l
`
`119
`
`234
`76
`
`11
`28
`13
`13
`1
`6
`2
`3
`19
`406
`
`•One or more of the following: cobalam·in malabsorption in part 1 of the
`Schilling test, achlorhydria, and autoimmune thyroid disease.
`tMalabsorption syndrome in a person who had Jived in the tropics (intestinal
`biopsy not performed).
`
`umented; all but one of these episodes showed at least
`one of the hematologic responses listed in Table II.
`fu five episodes showing a therapeutic response, pre(cid:173)
`treatment blood smears were normal or nondiagnos(cid:173)
`tic (marrow aspirates were not obtained). Of the 434
`episodes, 421 (970,,6) responses to treatment with
`cyanocobalamin were documented (Table II).
`Serum metabolites were measured in 123 episodes
`of folate deficiency diagnosed in 119 patients (four
`patients had two episodes). Our definition of folate
`deficiency was based on the clinical, morphologic,
`and biochemical criteria sununarized in Table Ill
`Hematologic responses to treatment with folic acid
`were documented for 94 episodes; adequate hemato(cid:173)
`logic follow-up data were not obtained for the others.
`Patients were considered folate or cobalamin defi(cid:173)
`cient based solely on the stated criteria and not on the
`results of the metabolite assays. The majority of the pa(cid:173)
`tients (86%) were studied before 1986, when the
`metabolite assays became available, and the percent of
`patients with each deficiency with elevated metabloite
`levels was similar for patients who were evaluated be(cid:173)
`fore and after 1986.
`
`MCV = mean corpuscular volume; LDH = lactate dehydrogenase.
`*Mean age 49 ± 17 years in folate-deficient group and 66 ± 18 years in
`cobalamin-deficient patients.
`tT otal number of cases for which information was available; denominator =
`number of patients, for sex and ethnic background, and number of episodes
`for other items.
`:tLeukopenia = leukocyte count less than 4,000/µL for white and Latino
`patients and less than 3,000/µL for blacks; thrombocytopenia = platelet count
`Jess than 150 ,000/µL.
`§Consistent with those syndromes seen in cobalamin deficiency [26].
`Peripheral neuropathy, ataxia, and cerebral dysfunction were often seen in
`folate-deficient alcoholics.
`
`(Table IV). Pernicious anemia, demonstrated by
`serial Schilling tests or serum antibody to intrinsic
`factor, was established for 57.6% and considered
`likely for another 18. 7%. The serum folate level was
`less than 2.1 nglmL in 17 patients and between 2.1
`ng/mL and 3.9 ng/mL in 57 patients in the cobalarnin(cid:173)
`deficiency group. All 7 4 patients had one or more find(cid:173)
`ings of serum antibodies to intrinsic factor, malab(cid:173)
`so:rption of vitamin B12 in Schilling tests, or neurologic
`dysfunction responsive to cobalamin treatment. Of
`these 74 patients, 19 had proven or probable tropical
`sprue, and it is likely that some had folate deficiency
`along with cobalamin deficiency. The 19 cobalamin(cid:173)
`deficient patients for whom diagnostic evaluation
`was inadequate to determine the etiology of the defi(cid:173)
`ciency (Table IV) had serum folate levels greater than
`4 ngtmL in all cases. Alcoholism caused 87% of the
`episodes of folate deficiency (Table IV).
`
`CAUSES OF DEFICIENCY STATES
`Diagnostic studies were adequate to determine the
`etiology of cobalamin deficiency for most patients
`
`Characteristics of Episodes
`The clinical features of the cobalamin- and folate(cid:173)
`deficient patients are summarized in Table V. The
`
`March 1994 The American Journal of Medicine Volume 96
`
`241
`
`Teva – Fresenius
`Exhibit 1018-00003
`
`

`
`SERUM METABOLITES IN COBALAMIN AND FOLATE DEFICIENCY/ SAVAGE ET AL
`
`300,000
`
`100,000
`
`50.000
`
`0
`
`~ 10,000 I
`~
`
`5000
`
`1000
`500
`
`300,000
`
`100,000
`
`50,
`
`c:i !ii 10,000
`
`5000
`
`0 i
`~
`
`1000
`
`'
`
`'It
`
`'
`
`l
`I
`I
`I
`
`I I
`I
`I
`I
`'
`I
`1
`
`·' .
`
`II
`
`'
`
`..
`
`• • •
`• ••.
`•
`•
`• • •
`,. • • • ·-~ : .,_
`• -"Sit ·.ra.1-e.
`• \. r. • •
`•
`-
`• .. • ...
`•
`: .· . .\~~
`..•
`... ~
`o -LA.
`.... ". . ... . .. .
`""~·-="· . . .. .
`r~;'l·~ :-·:·:
`... ·"
`
`~1:' ~-" ... : •
`
`•
`
`•
`
`.....
`~---------------------------
`
`II
`
`20
`
`so
`
`100
`
`250
`200
`1so
`HOMOCYSTEINE, µmo!IL
`
`300 w~75
`
`·' I I
`
`•
`
`•
`
`• ••
`
`•
`
`•
`
`•
`
`• •
`•
`
`•
`
`• •
`•
`
`•
`
`•
`
`•
`
`I
`I
`I
`I
`I
`I
`I
`•
`I
`I
`.. • • •
`I •
`I
`... •
`I
`• •
`I
`• •
`• ..
`••
`·. .
`••
`'
`I
`-
`I
`•
`,,._._ ..
`~
`1:~·-:
`'f :);~.
`~-·
`~-~-----------------
`
`TABLE VI
`Serum Concentrations of Methylmalonic Acid and Total
`Homocyateine In Episodes of Cobalamln Deficiency With and
`Wlhout Anenn
`·
`Anemia Present' Anemia Absent All Episodes
`No. %
`No. %
`No. %
`313
`121
`434
`
`Total number of episodes
`Episodes with
`Elevated§ MMA
`308 98.4
`427 98.4
`119 98.3
`306 97.8t 110 90.9t 416 95.9
`Elevated§ HCYS
`Both MMA, HCYS elevated
`302 96.5t 108 89.3:1: 410 94.5
`MMA alone elevated
`1.9
`17
`6
`11
`9.2
`3.9
`1.3
`4
`2
`1.7
`HCYS alone elevated
`6
`1.4
`MMA. HCYS both normal
`1
`0.3
`0
`0
`0.2
`MMA = me1hyknalonic acid; HCYS = total homocysteine.
`*Hematocrit less than 40% in males; less than 35% in females. The mean
`corpuscular vokrne was normal (80 to 100 fl) in 9 .9% of the episodes when
`anemia was present and in 33.3% of those when anemia was absent
`tp <0.005.
`tp <0.01.
`§More than 3 SD above the mean in normal control subjects.
`
`TABlE VII
`Serum Metabolite Concentrations in Anemia Caised by Cobalamln
`or Folate Deficiency
`
`Total no. of episodes*
`Elevated MMA
`Elevated HCYS
`Both MMA, HCYS elevated
`Elevated MMA, normal HCYS
`Elevated HCYS, normal MMA
`MMA, HCYS both normal
`
`Cobalamin Deficiency
`No.
`%
`297
`292
`290
`286
`6
`4
`
`98.3
`97.6
`96.2
`2.0
`1.3
`0.3
`
`Folate Defici~
`%
`No.
`98
`4t
`88
`4
`0
`84
`10
`
`4.1
`89.8
`4.1
`0
`85.7
`10.2
`
`MMA = methyrnalonic acid; HCYS = total homocysteine.
`•Sixteen patients with cobalamin deficiency and 25 with folate deficiency with
`elevated serum creatinine levels were excluded from the analysis.
`tSevere volume depletion present in three episodes.
`substantial nwnbers of black and Latino patients with
`cobalamin deficiency reflect the distribution of eth(cid:173)
`nic groups in the catclunent areas of the two hospi(cid:173)
`tals and are consistent with previous reports of a high
`prevalence of pernicious anemia in these populations
`[32-34]. The higher proportion of black patients and
`anemic patients in the folate-deficient group cmne
`from including several series of alcoholics with ane(cid:173)
`mia studied at Harlem Hospit.al Center [23,25].
`The mean corpuscular volume (MCV) was normal
`in 17% of cobalamin-deficient patients arid in 25% of
`folate-<:leficient patients. Only a minority of patients
`with either deficiency state had leukopenia, pancy~
`topenia, atrophic glossitis, or marked elevations of
`serum lactate dehydrogenase. More than a third of the
`cobalamin-deficient group has neuropsychiatric
`symptoms and signs (Table V) compatible with that
`vitamin deficiency [26]. Peripheral neuropathy, ataxia,
`and cerebral dysfunction were frequently associated
`with alcoholism in the folate-deficient patients.
`
`20
`
`50
`
`150
`100
`HOMOCYSTEINE, µmolA.
`
`200 220
`
`Fipre 1 Top. Serum concentrations of methylmalonic acid and
`total homocysteine in 313 episodes of megaloblastic anemia due
`to cobalarn·in deficiency. Dashed tines indicate three standard
`deviations above the mean for normal controls for each metabolite;
`hatched area includes values for both metabolites less than three
`standard deviations above the mean for normal controls; open
`circles indicate 16 cobalamin deficiency episodes associated with
`anemia with elevated serum creatinine.
`Figure 1 Bottom. Serum concentrations of methylmalonic acid
`and total homocysteine in 121 episodes of cobalamin deficiency in
`which the hematocrit was normal (greater than or equal to 40% in
`men, greater than or equal to 35% in women). Dashed lines
`indicate three standard deviations above the mean for normal
`controls for each metabolite; hatched area includes values for
`both metabolites Jess than three standard deviations above the
`mean for normal controls.
`
`242 March 1994 The American Journal of Medicine Volume 96
`
`Teva – Fresenius
`Exhibit 1018-00004
`
`

`
`SERUM METABOLITES IN COBALAMIN AND FOLATE DEFICIENCY/ SAVAGE ET AL
`
`1000
`
`500
`
`I l e
`t 0
`
`0
`
`0
`
`0
`
`..,_
`•••'•A .•
`
`. . . . , _ . 0
`0
`•
`0
`
`:
`
`a
`o
`o
`0
`0
`J_~.-.:,-6:"-----------------
`•
`0
`. , ' ' · 0
`•
`• • • • ,,,..
`0
`
`Serum Metabolites in Cobalamin Deficiency
`Serum methylmalonic acid levels were markedly
`increased (more than 3 SD above the mean in normal
`controls) in 427 (98.4%) of the 434 episodes of cobal(cid:173)
`amin deficiency (Figure 1, Table VI); serum total
`homocysteine concentrations were markedly in(cid:173)
`creased (more than 3 SD above the mean in normal
`controls) in 416 (95.g<>,t,). fu 410 episodes (94.5%), both
`metabolite levels were markedly elevated. fu only one
`cobalarrrln-deficient patient (0.2%) were both metabo(cid:173)
`lites normal.
`The hematocrit was normal (Figure 1 Bottom; Table
`VI) in 121 of the 434 episodes of cobalamin deficiency.
`Nonetheless, serum methylmalonic acid values were
`elevated in 98.3% of the episodes without anemia fu
`contrast, homocysteine levels were elevated in 90.9%
`of the nonanemic patients compared with 97.8% of
`anemic patients (Table VI; chi squared = 8.66,
`p <0.005). Thus serum methyhnalonic acid levels were
`more sensitive for identifying nonanemic cobalamin(cid:173)
`deficient patients than serum homocysteine levels
`(p <0.005). Both metabolites were elevated in 89.3%
`of the episodes in which the hematocrit was normal.
`Mean homocysteine levels were significantly higher in
`anemic than nonanemic patients (89.4 ± 55.0 µmol/L
`versus 60.2 ± 41.3 µmol/L, p <0.001). Mean methyl(cid:173)
`malonic acid concentrations were also higher in ane(cid:173)
`mic patients, but not significantly so (14,663 ± 30,698
`nmol/L versus 8,599 ± 25,554 nmol/L, p >0.05).
`The clinical, hematologic, and neurologic features
`of the 18 episodes with normal serum homocysteine
`levels did not differ from those of the 416 with ele(cid:173)
`vated levels, except that anemia was generally less
`severe when they were normal (mean hematocrit,
`37.2% versus 28.3%, p <0.001). The seven patients with
`normal mefuylmalonic acid values did not differ sig(cid:173)
`nificantly in any respect from the 427 with elevated
`values.
`Serum creatinine was elevated (open circles,
`Figure 1 Top) in 16 cobalamin deficiency episodes,
`all of which were anemic. The mean serum homo(cid:173)
`cysteine was higher in these 16 episodes than in the
`297 associated with anemia but with normal serum
`creatinine (139.5 ± 103.0 µmol/L versus 86. 7 ± 50.0
`µmol/L, p <0.001). Similarly, mean methylmalonic
`acid values were higher when megaloblastic anemia
`was associated with renal failure (36,527 ± 48,036
`rnnol/L versus 13,450 ± 29,098 nmoJ/L, p <0.005).
`Even though many of the serum specimens had
`been stored at -20°C for 10 to 20 years, there was no
`indication that methylmalonic acid or homocysteine
`levels varied inversely with increased storage time.
`For example, metabolite levels in serum obtained
`from cobalamin-deficient patients between 1968 and
`1973 did not differ significantly from those in serum
`
`.
`
`•
`
`•
`
`•
`
`•
`•
`
`••••
`•
`
`0
`
`•
`
`20
`
`50
`
`150
`100
`HOMOCYSTEINE, µmol/L
`
`200
`
`250
`
`Figure 2. Serum concentrations of methylmalonic acid and total
`homocysteine in 123 episodes of megaloblastic anemia due to
`folate deficiency. Open circles indicate patients with elevated
`serum creatinine concentrations; boxes indicate three patients with
`severe hypovolemia and normal creatinine levels. Dashed lines
`indicate three standard deviations above the mean for normal
`controls. (Note that the scale for methylmalonic acid values differs
`from that in Figure l.J
`
`obtained during the most recent 5 years of the study,
`1984 to 1989 (data not shown).
`In a series of 86 consecutively studied patients with
`cobalamin deficiency [4] tested by earlier assay meth(cid:173)
`ods, 12 (14%) did not have elevated methylmalonic
`acid and 13 (15.1%) did not have increased homo(cid:173)
`cysteine. Sera were available from most of these pa(cid:173)
`tients for assay by the modified techniques used in
`this study. Seven of 10 patients with methylmalonic
`acid levels less than 3 SD above the normal mean us(cid:173)
`ing the older method had elevated concentrations us(cid:173)
`ing the current assay system. Of 11 previously normal
`homocysteine values, four were increased when re-(cid:173)
`assayed by the modified method.
`
`Serum Metabolites in Folate Deficiency
`Serum homocysteine levels were markedly ele(cid:173)
`vated in 112 (91.0%) of the 123 episodes of folate de(cid:173)
`ficiency (Figure 2). The 11 patients with normal lev(cid:173)
`els did not differ from the others in severity of anemia,
`megaloblastic morphologic changes, methylmalonic
`acid elevation, time in hospital before study, reticu(cid:173)
`locyte counts, or serum levels of folate, cobalamin,
`creatinine, bilirubin, or albumin (data not shown).
`Serum methylmalonic acid was increased in 15
`(12.2%) of the 123 episodes of folate deficiency
`(Figure 2); however, 14 of these patients had renal
`dysfunction (11 with serum creatinine elevation and
`3 with clinical evidence of severe volume depletion).
`
`March 1994 The American Journal of Medicine Volume 96
`
`243
`
`Teva – Fresenius
`Exhibit 1018-00005
`
`

`
`SERUM METABOLITES IN COBALAMIN AND FOLATE DEFICIENCY/ SAVAGE ET Al
`
`Excluding these 14 patients leaves serum methyl(cid:173)
`malonic acid elevation in only one episode of folate
`deficiency; and in that patient, the increase was mod(cid:173)
`est (398 nM/L).
`
`Serum Metabolites in Cobalamin
`Versus Folate Deficiency
`The serum metabolite levels of the folate-deficient
`patients, all of whom were anemic, were compared
`with those of the cobalamin-deficient patients with
`anemia (Table VII). Patients with elevated serum
`creatinine values (16 with cobalamin deficiency and
`25 with folate deficiency) were excluded from the
`analysis. Serum methylmalonic acid was markedly el(cid:173)
`evated in 98.3% of the 297 cobalamin deficiency
`episodes and in only 4.1% of the 98 folate deficiency
`episodes (chi squared= 343, p <0.001). Three of the
`four patients with normal serum creatinine and ele(cid:173)
`vated serum methylmalonic acid had orthostatic hy(cid:173)
`potension due to severe volume depletion; therefore,
`only 1 episode of folate deficiency was associated
`with an unexplained (and presumably false-positive)
`increase in serum methylmalonic acid.
`Serum homocysteine was markedly elevated in
`97.6% of the cobalamin deficiency episodes and in
`89.SoAi of the folate deficiency episodes (chi squared =
`9.19, p <0.005). The mean homocysteine was higher
`(87.3 ± 50.1 µmol/L) in anemic patients with cobal(cid:173)
`amin deficiency than in those with folate deficiency
`(58.3 ± 37.7 µmol/L, p <0.001). Both serum methyl(cid:173)
`malonic acid and homocysteine were markedly ele(cid:173)
`vated in 96.20A> of the anemic cobalamin-deficient pa(cid:173)
`tients in contrast to 4.1% of the folate-deficient
`patients (chi squared= 316, p <0.001).
`Elevated serum homocysteine without increased
`serum methylmalonic acid occurred in 85. 7% of the
`episodes of folate deficiency but in only 1.3% of the
`episodes of cobalamin deficiency (Table VII).
`Therefore, in the 395 anemic patients with normal
`serum creatinine concentrations, the specificity of an
`isolated increase in serum homocysteine for diag(cid:173)
`nosing folate. deficiency as a cause of megaloblastic
`anemia was 98. 7%.
`
`Analysis of Findings Using a Conventional
`Definition of Normality
`The data were also examined using 2 SD above and
`below the mean for healthy controls to define nor(cid:173)
`mality (normal range for serum methylmalonic acid,
`73 to 271 nmol/L; and for serum homocyst.eine, 5.4 to
`16.2 µrnol/L). This analysis showed methylmalonic
`acid elevated in 98.So/ci and homocysteine in 99.1% of
`the 434 episodes of cobalamin deficiency. Of the 121
`episodes of folat.e deficiency, homocysteine was in(cid:173)
`creased in 97.5% and methyhnalonic acid was elevated
`in 30.9%.
`
`COMMENTS
`Concentrations of methylrnalonic acid and homo(cid:173)
`cYsteine are stable indefinitely in serum stored at
`-20°C [13,14], and we found no trend toward lower
`values in serum that had been repeatedly thawed and
`frozen over a period as long as 20 years. Hence, the
`stability of these compounds allowed us to test their
`diagnostic usefulness for a large series of patients with
`well-document.ed cobalamin and folate deficiency that
`would have required decades to assemble prospec(cid:173)
`tively. We included all patients with these deficiencies
`seen at two New York City hospitals between 1968 and
`1989 from whom serum was available, after excluding
`patients with low or borderline levels of both vitamins
`when it was uncertain which deficiency they had.
`The results point to the high sensitivity of serum
`methylmalonic acid and homocysteine concentra(cid:173)
`tions, using the modified assay methods, for diag(cid:173)
`nosing patients with clinically significant cobalamin
`deficiency. One or the other metabolite level was
`markedly elevat.ed (greater than 3 SD above the mean
`in normal controls) in all but one of the 434 episodes
`(Table VI, Figure 1), giving a combined sensitivity of
`99.So/ci for the metabolites for detecting deficiency of
`the vitamin.
`While methylmalonic acid and homocysteine con(cid:173)
`centrations were both increased in approximately
`98% of cobalamin-deficient patients with megaloblas(cid:173)
`tic anemia, the methylmalonic acid level was more
`consistently elevated in nonanemic patients (Table
`VI). Using the modified techniques, methylmalonic
`acid determinations also had greater sensitivity for
`detecting mild or early cobalamin deficiency in pa(cid:173)
`tients with pernicious anemia receiving infrequent
`maintenance therapy with cobalamin [6] as well as in
`elderly patients with low-normal serum cobalamin
`levels [35]. Moelby and colleagues also found serum
`methylmalonic acid elevated in 30 of 31 patients with
`cobalamin deficiency [12].
`In spite of the generally high sensitivity of serum
`methylmalonic acid determinations, concentrations
`were normal in 7 of 434 episodes of cobalamin defi(cid:173)
`ciency (Table VI); however, the homocysteine level
`was increased in six of the seven episodes, indicat(cid:173)
`ing that the two metabolite determinations combined
`improve the rate of detecting cobalamin deficiency
`over that of methyhnalonic acid alone. The reason pa(cid:173)
`tients occasionally fail to show a methyhnalonic acid
`elevation is not clear, although gut :flora alt.erations
`that disturb propionate production by ent.eric bacte(cid:173)
`ria may play a role [6].
`We have included in the cobalamin-deficient group
`7 4 patients whose serum folate levels were also low
`or at the lower end of the normal range. Most of these
`patients had underlying pernicious anemia, docu(cid:173)
`mented by serum antibodies to intrinsic factor, or
`
`244 March 1994 The American Journal of Medicine Volume 96
`
`Teva – Fresenius
`Exhibit 1018-00006
`
`

`
`SERUM METABOLITES IN COBALAMIN AND FOLATE DEFICIENCY/ SAVAGE ET AL
`
`cobalamin malabsorption corrected by intrinsic fac(cid:173)
`tor in Schilling tests pe:rformed many weeks after cor(cid:173)
`recting the deficiency state. In some of the patients
`with tropical sprue, however, folate deficiency may
`have coexisted with cobalamin deficiency, or con(cid:173)
`ceivably, may have been the primacy deficiency.
`Serum methylmalonic acid was elevated in 72 and
`serum homocysteine in all of the 74 patients consid(cid:173)
`ered cobalamin deficient who had low or low-normal
`folate values. Excluding these patients from the analy(cid:173)
`sis would not significantly change any of the per(cid:173)
`centages listed in Table VI-in fact, the percentage of
`patients with cobalamin deficiency and increased
`methylmalonic acid level would be even higher.
`Patients with folate deficiency would be expected
`to have a block in ,the conversion of homocysteine to
`methionine [14); and indeed, serum homocysteine
`was markedly increased in 91 % of the 123 episodes
`of folate deficiency, confi.mling earlier reports of
`smaller numbers of pat