Gagnon et al. September 1987 Am J Obstet Gynecol

We thank Professor G. S. Dawes and Mr. Isaac Ra­poport for their help with this project and Mrs. H. Cheung and Mrs. T. Clarke for their kind technical assistance.

REFERENCES 1. Devoe LD, Castillo RA, Sherline DM. The nonstress test

as a diagnostic test: a critical reappraisal. AM J 0BSTET GYNECOL 1985;152:1047.

2. Thacker SB, Berkelman RL. Assessing the diagnostic ac­curacy and efficacy of selected antepartum fetal surveil­lance techniques. Obstet Gynecol Surv 1986;41: 121.

3. Dawes GS, Visser GHA, Goodman JDS, Redman CWG. Numerical analysis of the human fetal heart rate: the qual­ity of ultrasound records. AM J 0BSTET GYNECOL 1981;141:43.

4. Dawes GS, Redman CWG, Smith JH. Improvements in the registration and analysis of fetal heart rate records at the bedside. Br J Obstet Gynaecoll985;92:317.

5. Gagnon R, Hunse C, Carmichael L, Fellows F, Patrick J. External vibroacoustic stimulation near term: fetal heart rate and heart rate variability responses. AM J OBSTET GYNECOL 1987;156:323-7.

6. Dalton KJ, Dawes GS, Patrick]. Diurnal, respiratory and other rhythms of fetal heart rate in lambs. AM J 0BSTET GYNECOL 1977;127:414.

7. Dawes GS, Houghton CRS, Redman CWG. Baseline in

human fetal heart rate records. Br J Obstet Gynaecol 1982;89:270.

8. Bailey BJR. Tables of the Bonferroni t statistic. J Am Statistical Assoc 1977;72:469.

9. Kleinbaum DG, Kupper LL. Applied regression analysis and other multivariable methods. Duxbury, North Sci­tuate: Duxbury Press, 1978:188.

10. Brown R, Patrick J. The nonstress test: how long is enough. AMJ 0BSTET GYNECOL 1981;141:646.

11. Campbell K, MacNeill I, Patrick]. Time series analysis of gross fetal body movements during the last 10 weeks of pregnancy. Ultrason Imaging 1981;3:330.

12. Dawes GS, Houghton CRS, Redman CWG, Visser GHA. Pattern of the normal human fetal heart rate. Br J Obstet Gynaecol 1982;89:276.

13. Patrick], Campbell K, Carmichael L, Probert C. Influence of maternal heart rate and gross fetal body movements on the daily pattern of fetal heart rate near term. AM J 0BSTET GYNECOL 1982;144:533.

14. Patrick], Carmichael L, Chess L, Staples C. Accelerations of the human fetal heart rate at 38 to 40 weeks' gestational age. AM j 0BSTET GYNECOL 1984; 148:35.

15. Dalton KH, Dawes GS, Patrick]. The autonomic nervous system and fetal heart rate variability. AM J 0BSTET Gv­NECOL 1983;146:456.

16. Wheeler T, Cooke E, Murrils A. Computer analysis of fetal heart rate variation during normal pregnancy. Br J Obstet Gynaecol 1979;86:186.

Riboflavin concentration in maternal and cord blood in human pregnancy

Nancy Wolfert Kirshenbaum, M.D., joseph Dancis, M.D., Mortimer Levitz, Ph.D., Jean Lehanka, B.S., and Bruce K. Young, M.D. New York, New York

Riboflavin concentration was measured in sera of a control population and in a series of paired maternal and cord sera. The assay technique was carefully validated and appears to be specific and reproducible. The mean riboflavin concentration in 12 apparently healthy adults was 116 ± 46 nmoi/L (SO). In 20 uneventful pregnancies the cord serum concentration was generally higher than the maternal concentration (158 ± 47 nmoi/L versus 113 ± 35 nmol/~; p = 0.001 ). The cord-to-maternal ratio in paired sera averaged 1.45 ± 0.44. There was no detectable difference in binding of riboflavin to cord and maternal serum proteins as measured by equilibrium dialysis (59.0% ± 17% versus 60.8% ± 16%). Comparison of protein binding by paired cord and mat_ernal sera yielded a ratio of 0.99 ± 0.13. The transplacental gradient of riboflavin concentration is unrelated to protein binding and is consistent with active transport by the placenta, as previously demonstrated in vitro. (AM J Oesrer GvNecoL 1987;157:748-52.)

Key words: Riboflavin concentration, maternal, cord, protein binding

From the Departments of Obstetrics and Gynecology and Pediatrics, New York University School of Medicine.

Presented at the Seventh Annual Meeting of The Society ofPerinatal Obstetricians, Lake Buena Vista, Florida, February 5-7, 1987.

Reprint requests:] oseph Dancis, M.D., New York University Medical Center, 550 First Ave., New York, NY 10016.

Riboflavin is an essential nutrient that must be deliv­ered to the fetus across the placenta. The human pla­centa, in vitro, actively transfers riboflavin from the maternal to the fetal circulation, establishing a gradient toward the fetus. 1

' 2 The situation in vivo is less certain.

748

Volume 157 Riboflavin concentration in maternal and cord blood 749 Number 3

Table I. Riboflavin concentration in maternal and cord blood at delivery, as reported in the literature

Gradient Reference Sample Maternal (nmol/L) Cord (nmol/L) (CIM)*

Baker et al.9

Lust et aJ.l0

Knobloch et al. 11

Clarke12

Blood Serum Blood Plasma

14 10 ll 3

675 ± 224 (SD) 15.4 ± 0.33 (SEM) 342 ± 3.40 (SD) 107

896 ± 238 59.9 ± 0.50 (SEM) 454 ± 10.9 (SD) 86

1.3 3.9 1.3 0.8

*The gradient is the ratio of cord-to-maternal serum riboflavin concentration.

Table II. Riboflavin concentration and protein binding in maternal and cord plasma

Patient

l:j: 2:j: 3:j: 4:j: 5:j: 6:j: 7:j: 8:j: 9:j:

lO:j: ll:j: 12:j: 13:j: 14:j: 15:j: 16 17 18 19 20

Mean± SD

Maternal (nmol/L)

103 104 204 138 128 89

106 92

114 95 73

114 107 63

100 154 188 102 Ill 76

113 ± 35

Cord (nmol/L)

189 153 260 154 139 185 189 94

171 137 81

234 246 121 106 139 161 135 135 140

158 ± 47

Gradient (CIM)*

1.8 1.5 1.3 l.l l.l 2.1 1.8 1.0 1.5 1.4 l.l 2.1 2.3 1.9 1.0 0.90 0.86 1.3 1.2 1.8

1.45 ± 0.44

Binding (CIM)t

0.76 0.82

0.93 0.96 1.0 0.96 l.l l.l 0.95

1.3

0.99 0.96 1.0

0.99 ± 0.13

The cord serum concentration is higher than the maternal concentration, with a p value of 0.001. Paired maternal and cord sera concentrations show, with few exceptions, a consistent difference that is not evident in protein binding.

*The gradient is the ratio of cord-to-maternal serum riboflavin concentration. tThe relative binding of riboflavin to cord and maternal serum was measured by equilibrium dialysis. :j:Vitamin supplement (3 mg/day riboflavin).

Previous studies of maternal and cord blood concen­trations have yielded widely disparate results probably because of differences in analytic technique (Table I).

In the present study the riboflavin concentration was measured in maternal and cord sera at the time of delivery by an analytic technique based on more re­cently developed methods. High-performance liquid chromatography provides a specificity that was not pre­viously possible, and analytic losses are corrected for by isotope dilution.

Material and methods

Riboflavin assay. The assays are performed in dim light because of the sensitivity of the riboflavin com­pounds. A blood sample is collected into a dry tube and permitted to clot on ice. It is centrifuged at 3000 rpm for 10 minutes and the serum is collected. The serum is

frozen at - 70° C until analyzed. Previous studies in our laboratory demonstrated that riboflavin, as identified by high-performance liquid chromatography, remains stable under these conditions for at least 7 months.

To the thawed sample add 0.1 ml 11C-riboflavin (approximately 2500 cpm) to 0.5 ml serum. Radioac­tivity is measured in triplicate in 50 fLl serum. Add 0.6 ml cold trichloroacetic acid, 20%, to the thawed sample and let stand at oo C for I0 minutes. Centri­fuge at 3000 rpm for 10 minutes, collect the superna­tant, and neutralize with 0.1 ml 2.4 mol/L K2HP04

buffer, pH 7.0. I~ect 0.5 ml supernatant onto high­performance liquid chromatography and fractionate as described below. The eluted fractions are scanned in the fluorometer. The radioactivity (in counts per min­ute per milliliter) and fluorescence are measured in the riboflavin peak.

750 Kirshenbaum et al. September 1987 Am J Obstet Gynecol

Recovery of radioactivity added to high-performance liquid chromatography was consistently >90%. Recov­ery of radioactivity through the entire analytic proce­dure varied from 85% to 90%.

Calculations Eluate (fluorescence)

~~~~--~~~~--~----- X (I)Riboflavin standard (fluorescence)

riboflavin standard (nmol/L) eluate concentration (A)

Correction for analytic losses Serum (cpm/ml) x Eluate (cpm/ml)

(2)

A = riboflavin concentration (serum + "C-riboflavin) (B)

Correction for added 14C-riboflavin serum (cpm/ml) ( )3

B - "C-riboflavin (cpm/ml) Riboflavin concentration (nmol/L)

The high-performance liquid chromatographic system has been described previously.' In this system, flavin adenine dinucleotide, flavin mononucleotide, and ri­boflavin elute at approximately 8.3, 9.3, and 10.3 min­utes, respectively. Fluorescence is measured on an Aminco-Bowman spectrofluorometer (American In­strument Co., Silver Spring, Maryland) with excitation at 463 nm and emission at 520 nm. Radioactivity is assayed in a Packard Tricarb scintillation counter (Pack­ard Instrument Co., Inc., Downers Grove, Illinois).

Material. Riboflavin, flavin mononucleotide, and flavin adenine dinucleotide were purchased from Sigma Chemical Co. (St. Louis, Mo.). The standard so­lutions are 26 nmol/L, 19.8 nmol/L, and 3.7 IJ.mol/L, respectively. They are kept shielded from light with aluminum foil at 4° C and are stable for at least 24 hours. "C-2-o-riboflavin (53 mCi/mmol) was purchased from Amersham (Arlington Heights, Illinois) . The pu­rity of the compounds was checked by paper chro­matography' and high-performance liquid chroma­tography.

Conversion to lumiflavin. Confirmation of the iden­tity of the riboflavin peak obtained from serum was sought by exposing it to photolysis and comparing its effect to that of similarly exposed authentic riboflavin. The serum and authentic riboflavin were subjected to high-performance liquid chromatography as described above. The riboflavin peak was identified by elution time and fluorescence. This fraction was lyophilized and redissolved in 0.5 ml water and approximately 1000 cpm "C-riboflavin was added. The specific activity was measured (in counts per minute per milliliter per fluorescence unit). The pH was raised to 8 to 8.5 with 4N KOH. The serum sample and the riboflavin stan­dard were transferred to glass tubes and simultaneously exposed to a I 50 W white light at I8 inches for 2 hours. The tubes were covered to prevent evaporation. The

pH was returned to neutral and 0.5 ml of each sample was rechromatographed on high-performance liquid chromatography, and the specific activity of the flavin derivative (elution time, I2.6 minutes) was measured.

Protein binding. Comparative binding of riboflavin by maternal and cord sera was measured by equilibrium dialysis. Paired samples of maternal and cord serum, 0.5 ml of each, were placed in individual dialysis bags and dialyzed together in a beaker against 10 ml Earle's buffered salt solution containing approximately 1000 cpm/ml 14C-riboflavin. The beakers were shaken gently for 5 hours at 37° C in the dark. The concentration of radioactivity in the bags and the medium was deter­mined at the end of incubation and percent binding was calculated.

Binding (%) = cpm/ml inside bag - cpm/ml outside OO

I . 'd X 1 cpmIm ms1 e Subjects. Laboratory personnel served as control

subjects for the determination of blood concentrations. Blood samples were drawn after a 2- to 3-hour fast. Only one subject (J. M. L.) took vitamin supplements.

Twenty mothers with uncomplicated pregnancies were selected for study. Mothers are routinely pre­scribed a multiple vitamin preparation (Stuartnatal, Stuart Pharmaceuticals, Wilmington, Delaware) at the first prenatal visit that contains a daily amount of 3 mg riboflavin. Five mothers were noncompliant and one patient (Table II, No. 15) discontinued supplementa­tion 1 month before delivery. The remaining patients took vitamin supplements until within 24 hours of de­livery. Blood samples were collected at delivery. Cord samples were drawn from double-clamped cord sec­tions. The blood was placed on ice and permitted to clot for I5 minutes. The serum was separated by cen­trifugation at 3000 rpm for 10 minutes and frozen at -70° C until analysis.

Results

The mean serum concentration of plasma riboflavin in 12 apparently healthy subjects on a freely selected diet was 116 ± 46 nmol/L (SD). Notable was the broad range (48 to 222 nmol/L) without evident symptoms.

Five assays were performed on one serum sample (J.D.) over a period of several weeks. The average SD was 8%. Five additional samples were assayed more than once. In four samples the SD was 10% or less and in the fifth it was I7%.

To further confirm the specificity of the assay, the riboflavin peak obtained by high-performance liquid chromatography of the serum sample was derivatized by photolysis, and its major fluorescent product, lu­miflavin, was measured as described in the Methods section. Six sera were studied. Fifty percent plus or minus seven percent of the riboflavin peak was recov­

Volume 157 Riboflavin conc~ntration in maternal and cord blood 751 Number 3

ered as lumiflavin after repeat high-performance liquid chromatography. In five sera the difference in lumi­flavin specific <J.Ctivity between authentic riboflavin and serum riboflavin was 8.6% ± 1.3% and in the sixth it was 20%. By this stringent test, >90% of the material in serum assayed as riboflavin was confirmed as ribo­flavin.

The plasma concentrations in the mothers at delivery were similar to those in the nonpregnant control populatiop (113 ± 35 nmol/L [SD] versus 116 ± 46 nmol/L), with no significant statistical difference be­tween the two groups. There wa~ no detectable effect of vitamin supplementation in the test series. The plasma concentration in the mothers who had taken supplements was 109 ± 32.4 nmol/L versus 130 ±

51.4 nmol/L in the mothers who had not taken sup­pl.ements (p = 0.87).

The cord plasma concentration was higher than the maternal concentration (158 ± 47 nmol/L [SD] versus 113 ± 35 nmol/L), with a p value <0.001 as deter­mined by the double-taikd Student t test. Examination of paired samples revealed a consistent gradient toward the fetus (18 of 20 samples). The mean gradient was 1.45 ± 0.44. Comparison of maternal vein and umbil­ical vein levels between vitamin-sqpplemented and nonsupplemented groups showed iqsignificant differ­ences in mean level <J.nd in the ratio of cord to maternal serum concentrations. The gradient was persistent and not significantly different for the supplemented versus unsupplemented groups. The ratio of the cord to the maternal serum concentration for the supplemented group was 1.54 ± 0.43, and for the unsupplemented group it was 1.23 ± 0.39.

Protein binding of riboflavin by maternal and cord plasma varied independently of the riboflavin concen­tration. The mean binding by 13 maternal plasmas was 60.8 ± 0.6 whereas that of the cord plasma was 59.0 ± 17. Inspection of the individual paired sera re­.sults confirmed the absenc~ of differential binding by m~ternal and cord sera (Table II).

Comment

Riboflavin, or vitamin B2 , is a water-soluble vitamin. It is an essential cofactor in cellular oxidation. It is present in the body as riboflavin and its related coen­zymes flavin adenine dinucleotide and flavin mono­nucleotide. Riboflavin is necessary for fetal growth and development. In experimental animals, riboflavin de­ficiency is associated with congenital malformations! Smithells et al.5 administered a multivitamin prepara­tion, including riboflavin, to pregnant women in a con­trolled study and found a significantly reduced inci­dence of neural tube defects ip the offspring.

Studies conducted in this laboratory by an in vitro perfusion technique demonstrated that riboflavin is ac­tively transferred across human placenta, establishing

a higher concentration in the fetal circulation than in the maternal circulation.1 The driving force is a very rapid uptake of riboflavin from the maternal circu­lation.•

Several factors in addition to placental performance affect the in vivo concentrations of riboflavin. Promi­nent among these are the rates of metabolism and ex­cretion of riboflavin by mother and fetus and binding to plasma protein. Therefore·, one can not assume from the in vitro studies where these factors were not op­erative that a similar gradient would be found in vivo.

Four reports were found in the literature in which maternal and cord blood concentrations were mea­sqred. For each investigat~on a different analytic tech­nique was used, and the results varied over a wide range (Table 1). In three reports the cord concentration was higher than the maternal concentration, and in the fourth the reverse was found. It appeared timely to apply some of the newer analytic techniques to this question. High-performance liquid chromatography was used to isolate riboflavin from other circulating fluorescent compounds. Isotope dilution was used to correct for analytic losses.

A control study was done on a series of 12 adults. The serum riboflavin concentration was 116 ± 46 nmol/L (SD). The range was broad (48 to 143 pmol/L). The concentration was higher than that reported by two previous ipvestigators using high­performance liquid chromatography. Ohkawa et a!." studied six subjects and reported a serum riboflavin concentration of 33 ± 2.2 nmol/L (SEM). Lambert et al.' reported a mean concentration of 25 nmol/L.

The following procedure was undertaken to confirm the specificity of our analytic technique. The riboflavin peak was collected after high-performance liquid chro­matography in the standard assay technique, alkalin­ized, and exposed to light. On rechromatography, the riboflavin peak had disappeared and a new fluorescent peak, presumably huniflavin, appeared, with an elution time of 12.6 minutes. By comparing the behavior of the serum "riboflavin" to authentic riboflavin, as de­scril:>ed in the Methods section, it was concluded that >90% of the serum riboflavin had been identified cor­rectly.

The analytic technique was used to measure the ma­ternal and cord serum concentrations in a series of 20 pregnancies (Table II). The riboflavin concentration in the cord was 158 ± 47 nmol/L (SD) as compared with 113 ± 35 nmol/L in maternal serum, a statistically sig­nificant difference (p = 0.00 1). The concentration gra­dient was confirmed by examining paired maternal and cord sera, the mean gradient being 1.45 ± 0.44. Long­term vitamin supplementation did not affect serum ri­boflavin levels and did not contribute to the maternal­fetal gradient established by dietary intake alone in this population.

752 Kirshenbaum et al.

The perfusion studies had demonstrated that human placenta is capable of establishing a gradient of this magnitude. However, it was possible that differential binding of riboflavin to maternal and cord serum pro­tein contributed to this effect. Specific binding of ri­boflavin to serum proteins has been reported.'·8 How­ever, direct measurement of riboflavin binding by ma­ternal and cord sera does not support the hypothesis. In maternal sera, 60.8% ± 16% (SD) of riboflavin was bound compared witq 59.0% ± 17% in cord sera. Ex­amination of the results from paired maternal and cord sera was confirmatory. The cord-to-maternal ratio of riboflavin binding was 0.99 ± 0.13 (Table II). It is clear that protein binding does not account for the trans­placental gradient.

The mean fetal: maternal concentration ratio in se­rum in this series ( 1.45) closely approximates that ob­served in vitro in perfusion studies ," suggesting that similar mechan~sms are operative. Synthesis of the in vitro and in vivo observations suggests the following description of the placental transport of riboflavin . Ri­boflavin is actively extracted from the maternal plasma, concentrated within the placenta, and transferred to the fetal circulation. The rate of transfer to the fetus exceeds the transfer rate in the reverse direction by a margin that satisfies fetal requirements and establishes a gradient.

REFERENCES 1. Dancis J, Lehanka J, Levitz M. Transfer of riboflavin by

the perfused human placenta. Pediatr Res 1985; 19: 1143-6.

September 1987 Am J Obstet Gynecol

2. Dancis J, Lehanka J, Levitz M, Schneider H . Establish­ment of gradients of riboflavin, L-lysine and a-aminoiso­butyric acid across the perfused human placenta. J Re­prod Med 1986;31:293-6.

3. Murthy CVR, Adiga PR. Isolation and characterization of a riboflavin-carrier protein from human pregnancy se­rum. Biochem Int 1952;5:289-96.

4. Warkany J, Nelson RC. Appearance of skeletal abnor­malities in the offspring of rats reared on a deficient diet. Science 1940;92:383-4.

5. Smithells RW, Sheppard SS, Seller MJ, et a!. Apparent prevention of neural tube defects by periconceptual vi­tamin supplementation. Arch Dis Child 1981;56:911-8.

6. Ohkawa H, Ohishi N, Yagi K. A simple method for mi­crodetermination of Havins in human serum and whole blood by high-performance liquid chromatography. Bio­chem Int 1982;4:187-94.

7. Lambert WE, Cammaert PM, Leenheer AP. Liquid chro­matographic measurement of riboflavin in serum and urine with isoriboflavin as internal standard. Clin Chern 1985;31 :1371-3.

8. Innis WSA, McCormack DB, Merrill AHJr. Variations in riboflavin by human plasma: identification of immuno­globulins as the major proteins responsible. Biochem Med 1985 ;34:151-65.

9. Baker H, Frank 0, DeAngelis B, Feingold S, Kaminetzky HA. Role of placenta in maternal-fetal vitamin transfer in humans. AM J 0BSTET GYNECOL 1981; 141:792-6.

10. LustJE, Hagerman PD, Villee CA. The transport of ri­boflavin by human placenta. J Clin Invest 1954;33:38-40.

11. Knobloch E, Hodr R, Janda J , Herzmann J, Hond­kova V. Spectroftuorometric micromethod for determin­ing riboflavin in the blood of newborn babies and their mothers. IntJ Vitam Nutr Res 1979;49:144-51.

12. Clarke HC. Distribution of riboflavin in blood: in women and prenates. IntJ Vitam Nutr Res 1977;47:361-3.