Laboratory Monitoring of Gestational Diabetes*

Transcription

1 ANNALS OF CLNCAL AND LABORATORY SCENCE, Vol. 21, No. 6 Copyright 1991, nstitute for Clinical Science, nc. Laboratory Monitoring of Gestational Diabetes* SHESHADR NARAYANAN, Ph D. Department of Pathology, New York Medical College/ Metropolitan Hospital Center New York, NY ABSTRACT The consequences of uncontrolled gestational diabetes is severe to both m aternal and fetal w ell-being. An ideal laboratory test to m onitor gestational diabetes should accurately reflect short-term glucose changes. Glycated album in, by virtue of its short half-life of 14 to 19 days, lends itself as a test to m onitor and control gestational diabetes. Analytical approaches for th e m easu rem ent of glycated album in are review ed, and the nonspecificity of the sim ple colorimetric fructosamine assay is stressed. The performance characteristics of fructosamine assay, and the merits of the one hour oral glucose screening test and three-hour oral glucose tolerance test are discussed. An objective strategy for laboratory monitoring of gestational diabetes would include other assays such as fasting plasm a glucose to correlate w ith glycated album in (fructosamine) and even to com plem ent the less sensitive glycated hem oglobin assay. ntroduction The developm ent of gestational-onset diabetes in the second half of pregnancy is more likely since placenta during that period produces progressively increasing amounts of estrogen, progesterone, and hum an chorionic som atom am m otropin also called hum an placental lactogen. Since these hormones are antagonistic to insulin, the pregnant m other develops insulin resistance leading to the onset of gestational diabetes. t is im portant that gestational-onset diabetes be brought u nder control, since otherw ise the condition can have grave consequences to both m other and the fetus. Since m aternal hyperglycem ia exposes the fetus to high levels of glucose, the fetal pancreas has to produce increasing amounts of insulin to m etabolize the increased glucose load. Since insulin promotes both fat and protein synthesis, the fetus may become so large as to cause complications (dystocia) during vaginal delivery, requiring del i v e r y b y c a e s a r e a n s e c t i o n in m any instances. A ddress re p rin t req u ests to Sheshadri N arayanan, Ph.D., D ep artm en t o f Pathology, N ew York M edical C o lle g e /M etro p o litan H o sp ital C e n te r, 1901 F irst A venue, N ew York, NY Diagnosis of G estational-onset D iabetes Gestational-onset diabetes is usually diagnosed in the second half of preg /91/ $01.50 nstitute for Clinical Science, nc.

2 LABORATORY MONTORNG OF GESTATONAL DABETES 393 nancy. T hose subjects w ho have not already been identified as having glucose intolerance before the 24th week of pregnancy should be screened. This is usually done betw een the 24th and 28th week of pregnancy. A oral glucose load of 50 grams is adm inistered any time during the day w ithout regard to the tim e of last meal. One hour later a blood sample is drawn, and if the plasma glucose level is greater than 140 milligrams per deciliter (mg per dl) or (7.8 mm per L-S.. Unit), the subject should b e given an oral glucose to leran ce test. T his is done by adm inistering an oral glucose load of 100 grams after a fasting blood specim en is drawn. Three separate blood specimens, each after one hour interval, should be drawn after the oral administration of glucose is complete. T he diagnosis of gestational-onset diabetes is m ade if two or more of plasma glucose values given below are m et or exceeded. Fasting: 105 mg per dl (5.9 mm per L), one hour after glucose load: 190 mg per dl (10.6 mm per L); two hours after glucose load: 165 mg per dl (9.2 mm per L); and three hours after glucose load: 145 mg per dl (8.1 mm per L).9 (Values in parenthesis are in S.., units). The g lu co se v a lu e s re p o rte d h e re w ere obtained w ith th e classical Smogyi N elson procedure. These values have been revised to reflect results that would be obtained w ith an oxygen uptake measurem ent-based glucose oxidase proced ure.2 U sing the corrected values subjects w ith a one hour plasm a glucose value of 135 mg per dl (7.5 mm per L) or greater after a 50 gram oral glucose load will receive the three-hour oral glucose tolerance test. Two of the three following values should be m et or exceeded to m eet diagnostic criteria for gestational diabetes: Fasting: 95 mg per dl (5.3 mm per L); one hour after glucose load: 180 mg per dl (10 mm per L); two hour: 155 mg per dl (8.6 mm per L); three hour: 140 mg per dl (7.8 mm per L). Recent studies have utilized a one-hour glucose value after screening load as low as 130 mg per dl (7.2 mm per L) to institute a three-hour oral glucose tolerance test.24 Laboratory M onitoring of Gestational D iabetes An ideal test for m onitoring of gestational diabetes should not be affected by the subject not fasting or the tim e of day of blood collection. t should also mirror short term changes in glucose levels. n th is re g a rd, g ly c a te d h e m o g lo b in (HbAiC) is too insensitive, since the life tim e of erythrocyte is as long as 120 days. As such, the glycated hem oglobin value will include in its m easurem ent window a period of normoglycemia prior to the onset of gestational diabetes. Because of this lim itation, m ethodology and performance characteristics for H b A ^ are not discussed in this paper. Suffice it to say, that two of the major m ethods for quantita tin g g ly c a te d h e m o g lo b in, (io n exchange chrom atography and affinity c h ro m a to g ra p h y ), e a c h h a v e th e ir own lim itations. Bearing in m ind that nearly half the glycation in hem oglobin is N-term inal, protein glycated solely at e-lysine groups with a pka of 10 to 11 will be fractionated with the unglycated protein, and, as such, the full extent of hemoglobin glycation w ill be u n d e re stim a te d. As is w ell known, Schiff base, carbam ylated hem o globin, and abnormal hemoglobins such as hem oglobin F w ill com igrate w ith HbAjC on ion exchange chromatography. n contrast, affinity chromatography of glycated hemoglobin on phenylboronate resins is much less sensitive to ph and te m p e r a tu r e v a r ia tio n s th a n io n - exchange chrom atography and Schiff base and abnorm al h em o g lo b in s do n ot in te rfe re, y et p ro te in s g ly cated w ith p h o s p h o ry la te d su g a r d e riv a tiv es are re ta in e d w eakly, and, are thus underestim ated.6

3 394 NARAYANAN Glycated album in, how ever, perm its m onitoring of short term changes in glucose levels since album in has a half-life of 14 to 19 days and has greater relevance to the discussion of gestational-onset diabetes. Laboratory m ethods for the m easurem ent of glycated album in are based on the m easurem ent of the fructosam ine m oiety w h ich re su lts w h en glucose undergoes a non-enzymatic reaction with protein, which usually is album in. An interm ediate labile compound, an aldim ine, or Schiff base is form ed which undergoes rearrangem ent (amadori rearrangem ent) to give a stable ketoam ine or fructosam ine.1,6,21 This non enzymatic glycation is dependent both on the tim e and the concentration of glucose, with the sugars glycating N -term inal am ino acids or side chain lysine groups. T he ketoam ine that is form ed is structurally a beta ketosylamine, whose ring structure is primarily in the pyranosyl form, although it does exist also in the furanosyl form.6 Approaches to M easurem ent of Fructosamine N i t r o b l u e T e t r a z o l i u m D y e R e d u c t i o n T he m ost popular approach that has b e e n adapted to autom ation on several clinical chemistry analyzers is based on the reduction of nitrobluetetrazolium dye (NBT) by fructosam ine under alkaline conditions. The basis of the assay is the ability of fructosamine to enolize rapidly under alkaline conditions to an eneaminol. The latter probably dehydrates to form a strongly reducing enediol, splitting off in the process the sugar-free protein moiety. t is this enediol that reduces NBT, and the resulting blue compound (formazan) is quantitated spectrophotom etrically (figure 1). The intensity of color formed in the reaction is dependant upon ph, tem perature, NBT concentration and quantity of serum used in the assay. Standardization of variables is the key to the optimization of the assay which clearly has no end point, since the alkaline conditions promote the reaction to proceed indefinitely, by converting the enediol oxidation product resulting from NBT reduction to a deep blue-black chromogen.6 Although the exact mechanism of the reaction is still not quite well understood, it is likely that superoxide radicals are involved.21 n a typical assay, serum is added to NBT reagent in buffer (carbonate buffer), at an optim ized ph of at S T ^.1 Absorbance at 530 nm is m easured generally 10 and 15 m inutes after initiation of assay. The rationale for making a m easurem ent after 10 m inutes is to minimize CH2-NH-PROTEN C = 0 HOCH c h 2oh ALKAL CH-NH-PROTEN C = 0 -NH-, PROTEN^ HOCH CH20H CHOH CHOH NBT HOCH ALKAL CHjOH CHROMOGEN ABSORBANCE AT 530 NM FRUCTOSAMNE ENEAMNOL ENEDOL F ig u r e 1. N itroblue tetrazolium dye (NBT) reduction by fructosam ine.

4 LABORATORY M ONTORNG OF GESTATONAL DABETES 395 the effect of interfering substances that are reported to reduce NBT during the first 10 m inutes of the reaction tim e.8 Even so, the assay has been adapted to use on analyzers w here the incubation tim e is only seven to eight minutes. A correction for album in concentration is reported to increase the sensitivity of the m easurem ent.26 T here is considerable controversy w ith regard to the matrix effects of the standards that are used.1 Originally 1-deoxy-l-m orpholinofructose (DMF) w hich is a ketoam ine was used as a standard in a 40 g per liter solution of album in.1 Subsequently, matrix effects, depending on w hether one uses D M F standards m ade up in serum or standards m ade up in album in, have b een ad d ressed as w ell as variations owing to batch to batch differences of album in.1 One approach to internal standardization is to assay the same serum sam ple tw ice, one aliquot w ith added D M F and the other without DM F.7 However, even this approach has b een questioned.4 The fructosam ine assay, w hen compared to the specific m easurem ent of glycated am ino acid residues o f hydrolyzed serum proteins by HPLC, over estimates 10-fold the fructosamine concentration m serum. Recently it was dem onstrated by use of a secondary serum protein standard, calibrated w ith prim ary standards such as polylysine and glycated hum an serum album in, th a t values o b tain ed using D M F standards w ere indeed 10 times too high.20 The discrepancy was related to the D M F standardization being affected by variables such as ph, NBT concentration, and th e in stab ility of D M F standards.11 T he reference interval for fructosam ine of to mm per L obtained by use of serum protein standard calibrated w ith polylysine or glycated hum an serum album in is said to agree with the m ean glycation of three jimol per gram of protein determ ined by the furosine HpLC m ethod with e-aminofructosellysine as standard.20 nterferences in Fructosamine Assay. Although n eith er the Schiffbase interm e diate nor glucose below ph 11 interfere w ith the fructosam ine assay, carbonyl groups associated w ith collagen cross links could be a source of interference.6 Among reducing substances glutathione and uric acid have been known to interfe re.6 W hen a d ap ted to au tom ation, n u m ero u s in te rfe re n c e s h av e b e e n reported, some varying from one analyzer to another. Thus, while on one discrete analyzer in terferences w ere rep o rted w ith bilirubin, heparin, cysteine, ethylene diam ine tetraacetic acid (EDTA) and uric acid, on another discrete analyzer in ad d itio n to interferen ces by b iliru bin, heparin, and cysteine, hemoglobin and g lu ta th io n e w ere also re p o rte d to interfere.1 T he poor solubility of the diformazan form ed during the NBT reduction reaction is also a variable in the fructosamine assay.11 U pon solubilization of diformazan in an optim ized autom ated assay, it was noted that uric acid contributed significantly to the color, w hich was c o m p e n s a te d for by a m a th e m a tical correction.25 n addition to the interferences e n u m erated previously, some NBT reducing activity in serum has been attributed to non-specific substances other than fructosam ine. Such non-specific NBT reducing activity varied from serum to serum and was not reducible w ith sodium borohydride.19 t has been reported that some of this non-specific NBT red u cin g activity is due to the presence of a vitamin: p y rro lo q u in o lin e q u in o n e (P Q Q ).17 Since PQQ is not reduced by sodium borohydride, w hereas fructosam ine is reduced, a correction for non-specific fructosamine activity can be performed. The correction entails using two aliquots of serum to one of which sodium borohydride is added which should reduce only fructosamine, while to the other aliquot sodium borate is added which will leave both fructosam ine and non-specific PQ Q

5 396 NARAYANAN activity intact. After this preincubation step, acid is added; after neutralization, NBT assay is performed. The apparent fructosam ine activity m easured in the presence o f sodium borohydride is subtracted from total fructosam ine activity m easured in the serum aliquot incubated with sodium borate to yield the true fructosam ine activity.17 A nother in terferen ce th at has been reported is due to im m unoglobulin-a (ga), w hich, since it is also highly glycated, gives spuriously high fructosamine values at pathological concentrations.22 T h i o b a r b i t u r i c A c i d P r o c e d u r e Several variations of this procedure have been utilized.1,21 Suffice it to say that they all involve prolonged heating for at least eight hours at 115 C to convert the keatoam ine lin k ag e in th e fructosam ine m olecule to 5-hydroxymethyl furfural (5-HMF). The latter compound is then reacted with thiobarbituric acid for 30 to 50 m inutes, and the resulting color is m easured in a spectrophotom eter at 443 nm. Results are reported as fructose equivalents using fructose as standard. Glucose in the patient s sample is a major interferent and has to be rem oved either by dialysis or by precipitation of proteins in clu d in g fructosam ine w ith trichloroacetic acid prior to conversion to 5-HMF. A serum blank has to be included to compensate for non-specific fructosam ine a c tiv ity by re d u c in g th e tru e fru c to sam in e w ith sodium b o ro h y d rid e. Even with the various modifications that this assay has undergone in an effort to optimize the assay and reduce the analysis tim e, the procedure is still involved, and, as such, is n o t am en ab le to routine usage.1 F u r o s i n e P r o c e d u r e Furosine (epsilon N-(2-furoylmethyl)- L-lysine) is one of the products that results w hen the epsilon amino group of lysine in the protein m olecule that is bound to glucose to form fructosamine is hydrolyzed with six molar hydrochloric acid at 95 to 100 C for 18 hours. Furosine is then separated on reverse phase high p erfo rm an ce liq u id chrom ato g rap h y (HPLC) with phosphoric acid (7 mol per liter) as the m obile phase and quantified by dual U.V. wavelength m easurem ents at 254 and 280 nm.21 Although this procedure offers a specific m easurem ent of fructosam ine since Schiff base, enzym atically bound sugar, or collagen cross link com pounds do not interfere, the prolonged hydrolysis step coupled with the use of a HPLC instrum ent is the major stum bling block for its use in the routine clinical laboratory. A f f i n i t y C h r o m a t o g r a p h y This procedure is based on the ability of com pounds containing Cis-hydroxy groups, such as glycated proteins, to form a complex w ith boronic acid w hich is used as an affinity support. However, it should be borne in m ind that phosphorylated sugars are hindered from binding to boronate resins. As such, not all glycated species of a protein are estim ated by phenyl boronate affinity chrom atography. E sse n tia lly, th e p ro c e d u re involves application of plasma or serum diluted preferably w ith buffer to a colum n contain in g im m o b ilized m -am inophenyl boronic acid.12,13 T ypically an ammonium acetate buffer 0.25 m olar containing 0.05 m olar m agnesium c h lo rid e adjusted to a ph varying from ph to ph is used to elute non-glycated album in. G lycated album in is subsequently eluted w ith either sodium citrate buffer at acid ph (0.2 molar, ph 4.5),13 or sorbitol buffer containing 0.2 m olar sorbitol, 0.05 molar EDTA and 0.1 molar Tris adjusted to a ph of 8.8, the same ph as the buffer used to elute non-glycated album in.12 Albumin in both glycated and

6 LABORATORY MONTORNG OF GESTATONAL DABETES 397 non-glycated fractions can be m easured either by dye-binding techniques using brom ocresol green at 630 nm 13 or by im m u n o tu rb id im e try a t 340 n m.12 Results are reported as percent glycated album in. The affinity procedure can be performed m anually with commercially available colum n kits or using an autom ated HPLC apparatus. The procedure, since it is free from major interferences including glucose, and the fact that Schiff base dissociates during chromatography and as such does not interfere, is the procedure of choice. However, the manual m anipulations using kit-based p rocedures, or alternatively the unavailability of HPLC apparatus in many clinical laboratories, lim its the application of the affinity procedure to routine use. This leaves the laboratorian with little choice but to rely on the popular and sim ple procedure utilizing NBT dye reduction for the m easurem ent of fructosamine in spite of its lim itations. Oral Glucose Screening Test and G lucose Tolerance Test. How good is the one-hour 50 gram oral glucose test for screening gestational diabetes? f the study is applied to a population of gestational diabetes subjects 25 years and older, the one-hour 50 gram oral glucose screening test can achieve a sensitivity of 88 percen t and a specificity of 82 p ercen t.10 H ow ever, if screening is conducted based on the presence of one or more of the clinical history factors, such as a family history of diabetes, hypertensio n, w e ig h t g a in, or p r o te in u ria, although the specificity rises to 87 percent, the sensitivity drops to 62 percent, making the screening test inadequate.10 n a recent study, subjects with a high risk for gestational diabetes were chosen and w ere given an adequate carbohydrate diet (a m inim um of 300 g per day) three days prior to adm inistering a 50 g oral glucose screening load, w ith the subjects fasting from m idnight up to the time of collection of th e fasting blood specimen. Subjects with a one-hour plasm a glucose value of greater than or equal to 135 mg per dl (7.5 mm per L) glucose w ere adm inistered a 100 gram oral glucose tolerance test. Gestational diabetes was established based on either a decidedly abnormal 50 gram screening test or an abnormal 100 gram oral glucose tolerance test as discussed previously.2 n this study, 66 percent of subjects (10 out of 15) who developed gestational diabetes w ere diagnosed during the first h a lf of the pregnancy. n the first trim ester, six patients were diagnosed. Lowering the abnormal one-hour plasma glucose value from 135 mg per dl (7.5 mm per L) to 130 mg per dl (7.2 mm per L) allow ed an additional two patients to be diagnosed in the first trimester, improving the sensitivity from 70 percent to 91 percent, with the specificity dropping just to 88 percen t from 91 percent. Perform ance Characteristics o f Fructosam ine Assay. How good is the fructosam ine assay? How does it correlate with the 50 gram oral glucose screening test and the three-hour glucose tolerance test in the monitoring of gestational diabetes? A recent study sheds some light on this subject. n this study, 97 pregnant women at 26th to 28th week of gestation w ere follow ed over a period of n ine m onths.3 O f these 97 wom en, only 13 w ere diagnosed to have gestational diabetes based on established criteria.2 Statistically significant differences (P < 0.005) w ere found in the fasting, one-, two-, and th re e -h o u r glucose values b etw een diabetic and non-diabetic pregnant subjects. n contrast, w hen samples collected at base line venipuncture for the 100 gram glucose tolerance test w ere assayed for serum fructosam ine and glycated hem oglobin level (HbA1C), no statistically significant difference was noted betw een the diabetic and nondiabetic subjects for both of these tests3 (Table ). These results w ould question the usefulness of serum fructosam ine as a screen-

7 398 NARAYANAN TABLE Comparison of Results Obtained on Serum Fructosamlne and Glycated Hemoglobin (HbAiC) at Base Line Venipuncture for the 100 Gram Glucose Tolerance Test a Test: 13 Subjects with Gestational Diabetes 84 Non-diabetic Pregnant Subjects (Controls) Subjects at 26th to 28th week of pregnancy Fructosamlne (mm) 2.02 ± ± 0.02 HbAiC (% ) 4.42± ±0.3 3 hour glucose tolerance test. Mean glucose values n mg per dl; values in (mm per Liter) (S.l. Units) Fasting 1 hour 2 hour 3 hour 122 (6.8) 234 (13.0) 194 (10.8) 140 (7.8) 90 (5.0) 158 (8.8) 135 (7.5) 99 (5.5) a Adapted from Cefalu, W.T.. Prather, K.L.. et al: Diabetes Care P < P < P < P < ing test for gestational diabetes. H ow ever, during follow up studies of diabetic subjects at two-week intervals, there was a significant correlation betw een fasting blood glucose and serum fructosamine levels (r = 0.81; P < 0.001). As expected, th ere was no correlation b e tw e en glycated hem oglobin (HbA1C) and fasting glucose levels (r = 0.11). T hese data w ould suggest th at the fructosam ine assay reflects short-tim e glycem ic control, and, as such, may be useful in monitoring the glycemic state of the pregnant m other.3 n another study, serum fructosam ine levels correlated significantly with m ean plasma glucose levels over a one to three w eek interval, with the closest association found in the preceding w eek.15 Eighty-five percent of wom en with gestational diabetes in this study had peak serum fructosamine values that were over the normal reference interval. n these pregnant women, there was an 88 percen t chance of an abnorm al glucose tolerance test, post-partum, w hen fructosamine levels were greater than 3.2 mm p e r L. Patients who had th eir fructosamine levels reduced to less than 2.5 mm p e r L had few er obese babies and lower cord insulin and C-peptide levels as compared to neonates of mothers with higher fructosam ine concentrations.15 n discussing perform ance characteristics of a method, the reference intervals, clinical sensitivity and specificity, analytical characteristics such as precision, and interferences need to be delineated. Attempts to summarize some of the perform ance characteristics of th e fructosamine assay are shown in table. n term s of esta b lish in g referen ce intervals, it has been pointed out from a stu d y on 40 n o n -d ia b e tic p re g n a n t w om en th a t th e re is a larg e in te r in d iv id u al variation for plasm a fructosamine that comes in the way of adaptin g an o v e ra ll re fe re n c e ran g e for p r e g n a n t w o m e n.23 n th is stu d y, although the interrun precision at two levels (1.46 mm per L and 2.8 mm per L) of plasm a fructosam ine w ere sim ilar (C.V. of 4.8 percent), the reference range for plasma fructosamine was betw een 0.3 to 0.9 mm per L, with a mean of 0.53 mm per L. Reference ranges established for normal pregnant women vary from study to study.5,16 As noted earlier, reference intervals would also depend on the stan-

8 LABORATORY M ONTORNG O F GESTATONAL DABETES TABLE Performance Characteristics of Fructosamlne Assay Reference ntervals for Pregnant Women fln mmol per Liter) Number of Subjects Normal Non-diabetic 2. N 63 Sensitivity 8 6 % 3. nterferences Mean of (Minimum 0.3; maximum 0.9) Specificity Prevalence (Gestational Diabetes) 95 % 11 % Diabetic Glutathione, uric acid, carbonyl groups associated with collagen cross links, bilirubin, heparin, cysteine, ethylene diamine tetraacetic acid (EDTA), hemoglobin, pyrroloquinoline quinone (PQQ), and mmunoglobulin-a (ga) Literature Reference , 27 5, , 27 1, 6, 17, dards used for calibration.20 O verlap betw een normal and diabetic pregnant subjects have been reported (table ), reflecting the insensitivity of serum fructosamine as a screening test to detect gestational diabetics.5 As to sen sitiv ity and specificity, a recent critical assessm ent of current literature on the fructosamine assay in the diagnosis and control of diabetes in general lam ented on paucity of information on th e subject.27 O f 65 articles from literature that the authors evaluated, only seven of them stated the sensitivity and specificity of the test or provided data to perform such calculations. Even these studies had limitations to the extent that standards such as selection of patients w ere not clearly stated, and relating fructosam ine levels to p atie n t categories were not m utually blind to the extent that the diagnosis was known even before the test was perform ed.27 Even so, in spite of the numerous interferences and technical problem s associated w ith the procedure, w hich w ere noted previously, the fructosamine assay, although not useful as a screening test for gestational diabetes, has m erit in the follow-up of the m aternal glycem ic state.3,15 T he recent critical assessm ent of literature leaves much room for improvement, a n d th ro u g h v a lid a tio n o f th e fru c tosamine assay, to remove doubts in the minds of the laboratorian on the validity of the fructosam ine test.27 Choice o f Laboratory Procedures fo r M onitoring Gestational Diabetes. Given the fact th at glycated album in (fructosamine) m easures short term glucose changes, it is superior to glycated hem o globin, which because of the long life tim e of the erythrocyte (120 days) m easures a period of normoglycemia that may have preceded the onset of gestational diabetes. So the averaged glycated hem o globin value may be too insensitive to accurately reflect the state of glycemia in the pregnant individual. Although the affinity chromatography procedure for glycated album in or glycated protein offers specificity, th e lack

9 400 NARAYANAN of access to HPLC apparatus in many clinical laboratories limits its use on a routine basis. Although inter-assay C.V. of 6.4 percent and 5.8 percent have been reported for glycated album in and glycated protein, respectively, on a manual a ffin ity c h ro m a to g ra p h y p ro c e d u re, doubts have been cast on the ability to distinguish betw een gestational diabetes patients and normal controls based on these m easurem ents.18 As such, it would appear that the affinity chromatography p ro ced u re for glycated alb u m in and glycated pro tein req u ires furth er critical validation. Because of the lack of specificity of the convenient NBT reduction assay for fructosamine, it has lim ited usefulness as a screening test for gestational diabetes.19 n this context, neither the fructosamine assay nor the glycated album in and glycated protein affinity chrom atography assays should replace the one-hour 50 gram oral glucose screening test for gestational d iab e tes.18 H ow ever, the fructosamine assay is useful to m onitor the glycemic state in the mother, as judged by tests such as fasting blood glucose and m ean outpatient blood glucose especially in the third trim ester of pregnancy.3 Conclusions G lycated album in assessed by a m easurem ent of fructosamine lends itself to monitor and control gestational diabetes. For purposes of base line comparison and to assess im provem ent in the glycemic state of the pregnant diabetic, it could even serve as a com plem entary test to glycated hem oglobin, even though the latter is not quite sensitive to m irror short term changes in glycem ia.21 T he rationale is to see if there is a shift in both the glycated album in and the glycated hem o globin value com pared to a base line value, and to determ ine if this shift correlates with the level of fasting blood glucose in the subject, thus providing a stricter laboratory m onitoring of gestational diabetes. R eferences 1. Ar m BRUSTER, D. A.: F ructosam ine: Structure, analysis and clinical usefulness. C lin. C hem. 33: , C a r p e n t e r, M. W. an d C o u s t a n, D. R.: C riteria for screen in g for gestational diab etes. Am. J. O bstet. G ynecol. 144: , C e f a l u, W. T., P r a t h e r, K. L., C h e s t e r, D. L., W h e e l e r, C. J., B is w a s, M., and P e r - NOLL, M. L.: T otal serum glycosylated p roteins in detection an d m onitoring of gestational diabetes. D iabetes C are 23: , C h r o m y, V., B r e in e k, P., S k l e p k o v a, A., and VALENDNOVA, M.: Fructosam ine: M atrix p roblem s w ith standards b a se d on p ro tein m atrices. C lin. C hem. 35: , 1989 (Letter). 5. C o m t o i s, R., D e s j a r l a is, F., N g u y e n, M., and B e a u r e g a r d, H.: C linical usefulness o f estim ation o f serum fructosam ine concentration as screenin g test for gestational d iabetes. Am. J. O bstet. G ynecol. 260: , F u r t h, A. J.: M ethods for assaying nonenzym atic glycosylation. Anal. B iochem. 275: , H o w e y, J. E. A., B r o w n i n g, M. C., a n d F r a s e r, C. G.: Assay o f serum fructosam ine th a t m in im iz e s sta n d a rd iz a tio n a n d m atrix problem s: U se to assess com ponents o f b iological variation. C lin. C hem. 3 3 : , Jo h n s o n, R. N. and B a k e r, J. R.: T h e alkaline reducin g activity o f g ly cated serum pro tein s and its relev an ce to d iab etes m ellitu s. C lin. C hem. 32: , N a t i o n a l D i a b e t e s D a t a G r o u p : C lassificatio n and d iag n o sis o f d iab e tes m ellitu s and other categories o f glucose intolerance. D iab e tes 28: , O S u l l i v a n, J. D., M a h a n, C. M., C h a r l e s, D., a n d DANDROW, R. V.: S c r e e n in g c rite ria for h ig h -r isk g e s ta tio n a l d ia b e t ic p a tie n ts. A m. J. O b s te t. G y n e c o l. 226: , P h il l ip o u, G., S e a b o r n, C. J., an d P h i l l i p s, P. J.: R éévaluation o f th e fructosam ine reaction. C lin. C hem. 34: , R e e d, P., B h a t n a g a r, D., D h a r, H., and WlNOCOUR, P. H.: P recise m easu rem en t o f glycated serum album in b y colum n affinity chrom ato g rap h y a n d im m u n o tu rb id im etry. C lin. C hem. Acta 262: , R e n d e l l, M., Ka o, G., M e c h e r i k u n n e l, P., P e t e r s o n, B., D u h a n e y, R., N ie r e n b e r g, J., R a s b o l d, K., Kl e n k, D., and S m i t h, P. K.: A m inophenylboronic acid affinity chrom atography and thiobarbituric acid colorim etry com p ared for m easu rin g glycated album in. C lin. C hem. 32: , 1985.

10 LABORATORY MONTORNG OF GESTATONAL DABETES R o b e r t s, A. B. a n d B a k e r, J. R.: Serum fructosam ine: A screenin g test for diab etes in pregnancy. Am. J. O b ste t G ynecol. 154: , R o b e r t s, A. B., B a k e r, J. R., Ja m e s, A. G., and H e n l e y, P.: F ructo sam in e in th e m anagem ent o f gestational d iab etes. Am. J. O bstet. G ynecol. 259:66 71, R o b e r t s, A. B., C o u r t, D. J., H e n l e y, P., B a k e r, J. R., J a m e s, A. G., a n d R o n a y n e,. D.: F ru c to sa m in e in d ia b e tic p regnan cy. L ancet ii: , R o s e n t h a l, M. A. an d G a l l o p, P. A.: E lim i nation o f n o n glycation d e riv e d in terferences in th e fructosam ine assay. C lin. C hem. 36:1114, 1990 (Abstract). 18. R y a n, E. A., S t a r k, R., C r o c k f o r d, P. M., an d S u t h i j u m r o o n, A.: A ssessm ent o f value of glycosylated alb u m in and pro tein in detection o f gestational d iab etes. D iabetes C are 20: , S c h l e i c h e r, E. D., M a y e r, R., W a g n e r, E. M., a n d G E R BT Z, K. D.: s seru m fru c to sam in e assay sp ecific for d eterm in atio n of glycated serum p ro tein? C lin. C hem. 34: , SCHLECHER, E. D. a n d V o g t, B. W.: S tandardization o f serum fructosam ine assays. C lin. C hem. 3 6 : , S c h l e i c h e r, E. D. and W i e l a n d, O. H.: Protein glycation: M easurem ent and clinical relevance. J. C lin. C hem. C lin. Biochem. 27: , S e g a d e -R o d r i g u e z, S., L o j o, S., C a m i n a, F. M., P a z, M., and D e l Ri o, R.: Effects o f various seru m p ro tein s on q u a n tifica tio n o f fructosam ine. C lin. C hem. 35: , S t a l e y, M. J. a n d M u r r a y -A r t h u r, F.: P lasm a fru cto sam in e in n o n -d ia b e tic p re g nancy. Brit. J. O b stet. G ynecol. 95: , S u p e r, D. M., E d e l b e r g, S. C., P h i l i p s o n, E. H., H e r t z, R. H., and Ka l h a n, S. C.: D iagn o sis o f g estatio n al d ia b e te s in e arly p re g nancy. D iabetes C are 24: , T a s, S. and Z e i n e l d i n, R. R.: A utom ated fructosam ine assay w ith im proved accuracy u se d to quantify nonenzym atic glycation o f serum proteins in d iab etes m ellitus and chronic ren al failure. C lin. C hem. 36: , V a n D ie ij e n -V i s s e r, M. P., S e y n a e v e, C., and BRUMBACHER, P. J.: n flu en ce o f variations in a lb u m in o r to ta l p ro te in c o n c e n tra tio n on serum fructosam ine concentration. C lin. C hem. 32:1610, (L etter) 27. W i n d e l e r, J. and Ko b b e r l i n g, J.: T h e fructosam ine assay in diagnosis and control o f diabetes m ellitus. (Scientific eviden ce for its clinical usefulness.) J. C lin. C hem. C lin. Biochem. 28: , 1990.