Human erythrocyte carbonic anhydrase I concentrations in dried blood spots from normal and hypothyroid neonates and children

Human Erythrocyte Carbonic Anhydrase I Concentrations in Dried Blood Spots from Normal and Hypothyroid Neonates and Children JEAN N. SHEPHERD and N. SPENCER

Department of Biochemistry, King's College, London WC2R 2LS England and J. A. HULSE The Hospital for Sick Children, London WC1 England The ELISA technique was used to assay carbonic anhydrase I (HCAI) in hemolysates prepared by the elution of dried blood samples from Guthrie cards. The ratio HCAI (mg)/hemoglobin (g) measured in blood samples eluted from Guthrie cards was not significantly different from that determined in aliquots of the same blood samples after storage as erythrocyte (RBC) lysates at -20°C, provided that the dried blood was eluted within three weeks of collection. The normal neonatal mean (SD) RBC HCAI concentrations were 2.05 (1.01) and 1.82 (0.86) mg HCAI/g hemoglobin for females and males respectively. Erythrocyte HCAI concentrations gradually rose with age, approaching normal adult levels by 16 years. Blood from hypothyroid neonates and hypothyroid infants on treatment gave normal HCAI/hemoglobin ratios.

KEY WORDS: carbonic anhydrase, erythrocyte, hypothyroidism, neonatology, reference values everal authors have reported a relationship between thyroid function and RBC HCAI concenStrations. In hyperthyroidism, RBC HCAI is significantly lower than normal in the vast majority of cases studied (1-7), whereas in hypothyroidism, the reported levels of RBC HCAI vary considerably. Magid (4) and Norgaard-Pedersen (7) reported that seven out of seven and ten out of eleven hypothyroid patients respectively had a higher than normal RBC HCAI concentration. Anker et al. (3) found that only three out of six hypothyroid patients had raised RBC HCAI levels and Auton et al. (2) assayed RBC HCAI in fifteen hypothyroid patients and found that in thirteen instances the levels were within, although at the upper end of, the normal range. Weatherall and McIntyre (5) assayed RBC HCAI in 8 hypothyroid patients and the level for each was higher than 2 S.D. from their normal mean. Twelve hypothyroid patients investigated by Funakoshi and Deutsch (6) had normal RBC HCAI concentrations. These discrepancies may be due to differences in assay techniques (8) or in the physiology of the patients. Most authors do not give the measure by which the patients were actually diagnosed as hypothyroid or the period of time since the condition was Correspondence: J. N. Shepherd, Department of Biochemistry, King's College, Strand, London WC2R 2LS, UK. Manuscript received February 2, 1984; revised manuscript received September 18, 1984; accepted for publication September 18, 1984. 62

severe enough to be clinically significant. Prolonged hypothyroidism may result in anemia (9) and elevated RBC HCAI levels have been reported in anemic states (10); however, in at least one of the above studies (4) where an increased RBC HCAI was reported in hypothyroidism, the patients were not anemic. Most of the above authors suggest that the measurement of RBC HCAI concentration may be useful in the diagnosis and assessment of hyperthyroidism and the same suggestion was made regarding hypothyroidism in those instances where raised HCAI concentrations were observed. Where measured, RBC HCAI concentrations returned to normal following treatment in both hyper- and hypothyroidism. The clinical value of RBC HCAI measurements in the treatment of thyroid disease remains uncertain. Congenital hypothyroidism occurs at an incidence of approximately 1 in 4000 births (11) and it has become increasingly common to screen newborn populations for this condition. For this purpose TSH is quantified in eluates from filter paper discs punched from dried blood spots which have been collected onto Guthrie cards. If there were a definite relationship between hypothyroidism and RBC HCAI concentrations, then the assay of RBC HCAI by ELISA might provide an alternative means of detecting hypothyroidism in the neonate or monitoring treatment. In this study we investigated the feasibility of applying the ELISA technique to the measurement of RBC HCAI (8) in samples collected onto Guthrie cards. HCAI concentration was measured on dried blood samples collected from normal neonates as part of a routine screening programme in order to establish a normal range. The enzyme was then assayed on dried blood samples collected from hypothyroid neonates. We also established a normal range ofRBC HCAI in children aged 0 - 1 6 years, and assessed the usefulness of RBC HCAI measurements in monitoring treatmen~ of hypothyroidism.

Materials The materials used for ELISA were as previously described (8). All general reagents were purchased CLINICAL BIOCHEMISTRY,VOLUME 18, FEBRUARY 1985

BLOOD CARBONIC ANHYDRASE I IN NORMAL AND HYPOTHYROID CHILDREN

from B.D.H. Chemical Co. U.K., or Sigma Chemical Co. Dried blood samples from normal and hypothyroid neonates were obtained from the Neonatal Screening Laboratory, The Hospital for Sick Children, Great 0rmond Street, London WC1. These were samples remaining after routine screening tests had been completed. Blood samples from "normal" infants and children up to 16 years were obtained from the Department of Haematology, The Hospital for Sick Children, Great 0rmond Street, London WC1. They were samples remaining after routine investigations had been completed. As far as possible the samples were chosen from patients undergoing minor operations. Blood samples from hypothyroid children on treatment were obtained from patients attending out-patient clinics. The patients cooperating in this study had all been diagnosed as hypothyroid from the regional screening programme and were receiving oral thyroxine therapy. Methods TREATMENT OF BLOOD SAMPLES

Blood from "normal" children was collected by finger prick but from hypothyroid patients by venepuncture. All samples were collected into EDTA and aliquots of the blood taken from hypothyroid patients were "spotted" onto Guthrie cards for a comparison of techniques. The plasma was removed from the remaining samples by centrifugation and the packed RBC washed three times with normal saline. During this process, care was taken not to remove any RBCs in case reticulocyteswere removed preferentially as this might inadvertently alter the HCAI/hemoglobin ratio in the RBC fraction. Washed erythrocytes were stored at -20°C for up to two months prior to assay. STORAGE AND ELUTION OF BLOOD SPOTS COLLECTED ONTO FILTER PAPER

Dried blood samples were stored at room temperature for no longer than three weeks; they were then eluted, as described below, and stored as lysates at -20°C for up to one month prior to assay. Discs (1/4" diameter) were punched from dried blood spots which had been collected onto blood collecting cards supplied by the Ministry of Health, U.K. (HMR 101/6, 17.8 mg/cm2). These discs were placed in small plastic tubes containing 150 mmol/L saline (1.0 mL) and left at 4°C overnight. Elution was aided by inverting the tubes two or three times during the first two hours.

2.5 to 25 ng HCAI/L; the solutions increased in concentration by increments of 2.5 ng/L. Hemolysates were diluted to give a hemoglobin concentration of approximately 1 g/L which was then accurately measured. These hemolysates were then further diluted 1:1000 with 0.5 mL/L Tween 20 in phosphate-buffered saline (PBS-Tween) before assay for HCAI. Hemolysates resulting from the elution of 1/4" diameter discs of dried blood invariably had a hemoglobin concentration close to 1 g/L. These hemolysates were, therefore, also diluted 1 : 1000 with PBS-Tween before assay. SEPARATION OF R B C ACCORDING TO CELL SPECIFIC GRAVITY

Erythrocytes were separated according to cell specific gravity using Ficol gradients (12). HORMONE ASSAYS

Radioimmunoassays were used to assay thyroid hormones (13, 14) and thyroid-stimulating hormone (11, 15). Results and discussion METHODOLOGY When 1/4 inch-diameter discs of filter paper containing dried blood were soaked in 150 mmol/L saline (1.0 mL) overnight at 4°C, hemoglobin was completely eluted. The HCAI concentrations of thirteen such eluates, when expressed as mg HCAI/g hemoglobin, were not significantly different from those found in aliquots of the same blood samples stored as packed RBC at -20°C, when the two groups of results were compared using Student's t-test (data not shown). However, after three weeks storage, the dried blood became difficult to elute from the paper and erroneous results were obtained. To avoid such difficulties all dried blood samples were eluted within three weeks of collection and stored at -20°C, as hemolysates, prior to assay. ERYTHROCYTEHCAI

CONCENTRATIONS

IN THE NEONATE

Hemoglobin concentration of dilute hemolysates was measured using Drabkins reagent.

Erythrocyte HCAI concentrations were measured in normal neonatal dried blood samples taken from 70 female and 71 male infants aged between six and ten days. The mean and standard deviation for each group were 2.05 -- 1.01 and 1.82 -+ 0.86 mg HCAI/g hemoglobin for females and males respectively. There was no significant difference between the values found in these two groups and in both instances histograms demonstrated normal distribution patterns. Combination of RBC HCAI values found for both male and female neonates provided a normal mean value of 1.93 -+ 0.93 (S.D.) mg HCAI/g hemoglobin.

ASSAY OF R B C H C A I BY E L I S A

ERYTHROCYTEHCAI CONCENTRATIONS IN NORMAL

MEASUREMENT OF HEMOGLOBIN

The conditions of assay were as previously described (8) except for the following minor modifications:Reference HCAI solutions were prepared containing CLINICALBIOCHEMISTRY,VOLUME 18, FEBRUARY 1985

CHILDREN

Erythrocyte HCAI concentrations were measured in 108 normal children aged between two days and one 63

SHEPHERD, SPENCER AND HULSE

2O.

•

"*~ Figure 1 - Relationship between age ~i and erythrocyte HCAI concentration in children aged between 2 days and 16 years." Each point represents a single individual: lines represent linear regression of HCAI (mg/gHb) on age (years), with 95% confidence limits (2 × S.D.) for HCAI indicated.

°•

°

°

.o

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°%'°

et 4

~

MONTHS

~

Ib

i'2

~

It~

YEARS

TABLE 1 Erythrocyte HCAI in Blood Spots from Hypothyroid Neonates

Patient 1 2 3 4 5 6 7 8 9

Age (days) 10 7 9 7 7 7 14 7 6

Sex (F = female) (M = male)

Erythrocyte HCA mg/g hemoglobin normal range (mean -+ 1 S.D.) M neonate 1.82 -+ 0.86 F neonate 2.05 4- 1.01

Blood TSH mU/L (normal neonatal value - - less than 5 mU/L)

M F F F F F F F M

1.27 4.06 1.88 1.7 3.4 0.99 2.6 1.4 1.1

157 114 87 344 261 187 376 270 265

year. The data are summarised in Figure 1 and it can be seen t h a t RBC HCAI concentrations, expressed per g hemoglobin, increased markedly during this first year of life. Linear regression analysis demonstrated a correlation between this rise in RBC HCAI and age (r= 0.63, p < 0.001, slope = 6.2). Data for a second group of 152 normal children aged from one year to sixteen years (Figure 1) were similarly analysed; RBC HCAI concentrations continued to increase with age but the rate of increase was markedly lower (r = 0.37, p < 0.001, slope = 0.23). These results are in excellent agreement with those of Berfanstam (16), who measured total RBC HCA activity in full-term neonates and children up to 12 years of age.

those samples used for the diagnosis of hypothyroidism. All samples were assayed within three weeks of collection. The results are given in Table 1 and it can be seen t h a t RBC HCAI concentrations were normal. Of the newly diagnosed patients listed in Table 1, RBC HCAI was assayed in three cases just prior to commencement of treatment, to see whether the longer exposure to low plasma thyroid hormone levels had affected RBC HCAI concentrations. In all instances RBC HCAI levels remained within the normal range. ERYTHROCYTE HCAI CONCENTRATIONSIN HYPOTHYROID CHILDRENAFTERTHYROXINETREATMENT

Erythrocyte HC AI concentrations were measured in nine hypothyroid, neonatal blood samples t a k e n at the same time and collected onto the same Guthrie cards as

Blood samples were taken from sixteen children, aged between one month and six years eight months, all of whom had been receiving oral T4 as t r e a t m e n t for hypothyroidism. Erythrocyte HCAI concentrations were measured on each sample and in all but three cases plasma T4 (free and bound), T~ and TSH were also assayed. The results are given in Table 2. In eight of the samples TSH levels were above normal but in most

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CLINICAL BIOCHEMISTRY,VOLUME 18, FEBRUARY 1985

ERYTHROCYTE HCAI CONCENTRATIONSIN HYPOTHYROID NEONATES

BLOOD CARBONIC ANHYDRASE I IN NORMAL AND HYPOTHYROIDCHILDREN TABLE 2 Analysis of Blood Samples Taken From Hypothyroid Children on Treatment

Patient 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Age (years)

Sex

Erythrocyte HCAI mg/g hemoglobin

F M F F F M F F M F F M F F M F

9.9 8.5 1.8 9.8 6.2 9.6 7.0 11.1 8.9 7.3 7.1 6.7 8.3 3.4 13.5 9.9

10/12 3 6 4 4 1 3 1 3 1 2 1 1 2

1/12 8/12 3/12 4/12 1/12 5/12 9/12 1/12 3/12 6/12 4/12 5/12

Serum TSH *less than 5 mU/L

Plasma thyroxine (total) "70-170 nmol/L

Plasma thyroxine (free) *8.8-23.2 pmol/L

7.4 <1.0 27.0 <1.0 <1.0 1.7 4.7 7.2 28.8 <1.0 2.7 22.7 21.7 55.0 6.0 --

114 128 126 148 --124 105 158 130 163 96 155 123 162 --

42.7 31.0 19.0 34.0 --14.0 15.7 26.5 21.5 24.2 14.4 20.9 21.4 20.3 --

Plasma triiodothyronine total "1.2-'2.8 nmol/L 1.8 2.1 1.9 2.2 2.3 2.2 2.7 2.8 2.4 2.1 2.9 2.1 2.5

*Normal range. TABLE 3 Analysis of Blood Samples Taken from Hypothyroid Children Taken off Treatment* for One Week

Age Patient (years) 26 27 28 29 30

1 1 3/12 1 2/12 1 2/12 1 4/12

Sex

Erythrocyte HCAI mg/g hemoglobin

F F F F M

4.0 10.4 9.5 8.9 11.3

Serum TSH mU/L **lessthan 5 mU/L 57.4 >128 43.2 90.4

Plasma thyroxine (total) *'70-170 nmol/L

Plasma thyroxine (free) **8.8-23.2 pmol/L

Plasma triiodothyronine (total) *'1.2-2.8 nmol/L

34 7 47 39 0

10.0 1.5 6.8 1.7 0.8

1.4 0.6 3.2 1.2 0.3

*Thyroxine treatment was stopped four weeks prior to the taking of the above blood samples. Oral triiodothyronine was administered for the first three weeks of the period after which all treatment was withdrawn (17). **Normal range. cases T4 (free and bound) and T3 concentrations were within, although at the upper limit of, the normal range. Without exception, RBC HCAI concentrations were normal in each individual when compared to their relevant age range. HCAI was assayed in blood samples taken from five congenital hypothyroid children when they were reassessed at approximately 1 year of age after a brief period of treatment withdrawal (17). The results are given in Table 3 and show that in each case the child was confirmed as hypothyroid but RBC HCAI remained normal. The above result was not altogether unexpected because RBC HCAI concentrations remain more or less constant throughout the life span of the red blood cell (18) and one week without treatment would most likely be insufficient time to see any effect of low thyroid hormone levels on the HCAI concentration of the total red blood cell population. However, if T4 controls the level of RBC HCAI via the mechanisms of either translation or transcription, then an effect might be apparent in the youngest cells (i.e. reticulocytes) in the blood

samples t a k e n from patients after one week of treatm e n t withdrawal. This possibility was investigated, particularly since Magid (4) had demonstrated t h a t the effects of t r e a t m e n t of hypothyroidism were quicldy evident if HCAI was assayed in the youngest fraction of erythrocytes. Blood samples were taken from three normal children (aged 4 weeks, 3 years and 9 years), from five hypothyroid children on treatment and from three hypothyroid children after a period of treatment withdrawal. Erythrocytes from each sample were separated according to specific gravity using a Ficol gradient. Concentrations of HCAI were measured in those cells representing the youngest, oldest and middle-aged erythrocytes. The results are given in Table 4 and it can be seen t h a t in the normal child, RBC H C A r concentrations are stable throughout the life span of the cell. There was no significant difference between HCAI concentrations in young and old erythrocytes in hypothyroid patients on or off treatment and it was not possible to demonstrate a relationship between low plasma thyroid hormone concentrations and the levels

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SHEPHERD, SPENCER AND HULSE TABLE 4 Measurement of HCAI in Erythrocytes of Different Ages Taken from Normal and Hypothyroid Children Erythrocyte HCAI m g / g hemoglobin Sample N1 N2 N3 23 24 20 21 22 22 26 29

Age of donor 4 3 9 1

weeks years years 6/12 years

1 2 1 1

9/12 1/12 3/12 3/12

years years years years

1 2/12 years

Clinical details Normal Normal Normal Hypothyroid Hypothyroid Hypothyroid Hypothyroid Hypothyroid Hypothyroid Hypothyroid Hypothyroid

-

on treatment on treatment on treatment on treatment on treatment off treatment off treatment off treatment

Unfractionated cells

New cells

Middle-aged cells

Old cells

3.4 13.5 6.0 6.4 10.4 7.2 9.5 8.9

3.8 7.9 7.8 3.4 14.9 5.4 7.5 10.9 7.3 10.6 9.4

3.2 7.2 7.0 2.8 12.7 5.6 6.2 11.9 6.7 9.7 9.0

3.5 7.9 7.9 3.1 13.9 5•5 6.6 11.8 7.1 10.6 8.5

Note: On treatment = oral thyroxine daily. Off treatment (see Table 3).

of H C A I in e r y t h r o c y t e s t a k e n from h y p o t h y r o i d children. T h e r e s u l t w a s n o t p r e d i c t a b l e from p r e v i o u s l y p u b l i s h e d d a t a a n d r a i s e d t h e i n t e r e s t i n g q u e s t i o n as to w h e t h e r RBC H C A I c o n c e n t r a t i o n s are, in fact, i n f l u e n c e d b y t h y r o i d function. O n e m u s t conclude from t h e e x p e r i m e n t s r e p o r t e d in t h i s p a p e r t h a t , in o u r h a n d s , RBC H C A I m e a s u r e m e n t s w e r e n o t u s e f u l as e i t h e r a m e a n s of d i a g n o s i n g h y p o t h y r o i d i s m or m o n i t o r i n g t r e a t m e n t . T h e r e s u l t s s u g g e s t t h a t t h e r e is no r e l a t i o n s h i p b e t w e e n low p l a s m a t h y r o i d h o r m o n e l e v e l s a n d RBC H C A I conc e n t r a t i o n s in t h e h y p o t h y r o i d child, a l t h o u g h t h i s w o u l d n e e d to be c o n f i r m e d b y t h e s t u d y of l o n g - t e r m h y p o t h y r o i d c h i l d r e n p r i o r to t r e a t m e n t .

Acknowledgements This project was supported financially by The Wellcome Trust Foundation. We thank Dr. B. G. Grant and Dr. N. Macintosh for permission to investigate their patients and Professor M. Hjelm for providing neonatal screening blood samples. Dr. B. Thompson and Mrs. D. Jackson assayed serum thyroid hormone and blood TSH levels respectively. The invaluable assistance of Mr. R. Brock and Dr. D. Bidwell is gratefully acknowledged.

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disorders? Scand J Clin Lab Invest 1970; 26: 257-62. 5. Weatherall DJ, McIntyre PA. Developmental and acquired variations in erythrocyte carbonic anhydrase isozymes. Brit J Haemat 1967; 13: 106-14. 6. Funakoshi S, Deutsch HF. Human carbonic anhydrases. v. levels in erythrocytes in various states. J Lab Clin Med 1971; 77: 39-45. 7. Norgaard-Pederson B, Lindholm J. Quantitation of red cell carbonic anhydrase B and C and fetal hemoglobin in thyroid disorders. Acta Med Scand 1972; 192: 227-30. 8. Shepherd JN, Spencer N, Bidwell DE, Voller A. The measurement of human erythrocyte carbonic anhydrase I by the enzyme-linked immunosorbent assay (ELISA). Clin Biochem 1982; 15: 248-51. 9. In: Kelley VC, Ed. Metabolic, endocrine and genetic disorders of children. Vol. I. p. 421. London, New York: Harper and Row, 1974. 10. Mondrup M, Anker N. Carbonic anhydrase isoenzyme B in erythrocytes of subjects with different types of anemia. Clin Chim Acta 1976; 69: 463-9. 11. Hulse JA, Grant DB, Clayton BE, Lilly P, Jackson D, Spracklan A, Edwards RWH, Nurse D. Population screening for congenital hypothyroidism. Br Med J 1979; 280: 675-8. 12. Fisher RA, Turner BM, Dorkin HL, Harris H. Studies on human erythrocyte inorganic pyrophosphatase. Ann Hum Genet 1974; 37: 341-6. 13. Amersham International Ltd., Data accompanying free thyroxine kit. 14. Morgans ME, Thompson BD, Whitehouse SA. Sporadic non-toxic goitre: An investigation of the hypothalamicpituitary-thyroid axis. Clin Endocrinol 1978; 8: 101-8. 15. Jackson DB, Vanderschueren-Lodeweyckx M, Grant DB. Thyrotrophin estimation in diagnosis and treatment of childhood thyroid disorders. Arch Dis Child 1975; 50: 522-5. 16. Berfanstam R. Studies on carbonic anhydrase activity in children. Acta Ped 1952; 41: 32-52. 17. Hulse JA, Grant DB, Jackson D, Clayton BE. Growth, development and reassessment of hypothyroid infants diagnosed by screening. Br Med J 1982; 284: 1435-7. 18. Funakoshi S, Deutsch HF. Human Carbonic Anhydrases. VI. Levels of isozymes in old and young erythrocytes and in various tissues. J Biol Chem 1971; 246: 1088-92. CLINICAL BIOCHEMISTRY, VOLUME 18, FEBRUARY 1985