FAST GLYCOSYLATION OF HÆMOGLOBIN

1142

pethidine blood concentrations did eventually approach "analgesic" levels in some patients by 30-40 min post injection. This resulted in no increase in analgesia and a transistory (10-15 min) mild sedation. In two patients, intravenous injection of naloxone 0.4 mg was followed by immediate reversal of sedation, with no change in level of analgesia: 30 min later there was a reduction but not a reversal of analgesic effect. These data strongly suggest that the initial analgesic effect of the high doses of epidural pethidine was due to a spinal action. At later stages (40-60 min post injection) analgesia may have resulted from a combination of a spinal action and the central-nervous-system effects associated with blood-borne pethidine. However, the reversal of sedative effects of pethidine by intravenous naloxone with no immediate change in analgesic effect favours the predominant action being on the spinal cord. Yaksh and Rudy have shown that naloxone administered directly into the C.S.F. rapidly antagonises the analgesic effect of subarachnoid morphine in animals. The lag-time between naloxone administration and antagonism of analgesia in our studies was probably due to the slow rate of naloxone delivery caused by the lower perfusion of the spinal cord compared to the brain. Neurological examination before and after epidural pethidine revealed no detectable change in sensory, motor, or sympathetic function. Absence of motor blockade was further illustrated by the ability of patients to ambulate within 30 min of injection. Absence of sympathetic block was confirmed by After 100 mg doses,

retention of

a

normal cobalt-blue

sweat

testS and absence of

postural hypotension after epidural pethidine. Venous occlusion skin plethysmography in the feet showed no evidence of increased blood-flow, and a normal ice responses was retained. In contrast

subsequent injection of 10 ml of local anaesthetic

resulted in at least a four-fold increase in abolition of the response to ice, indicating complete sympathetic blockade in lower limbs. Although the local anaesthetic injection was effective in relieving pain, this was accompanied by sympathetic, sensory, and motor blockade. None of the patients with chronic pain obtained complete pain relief with a control injection of saline whereas all obtained complete pain relief with epidural pethidine. These pharmacokinetic and neurological data provide strong support for a selective spinal analgesic action of epidural pethidine in man. The absence of changes in sensory, sympathetic, and motor function indicates that this form of analgesia may have considerable advantages for relief of severe acute and chronic pain in man.

(0.5% bupivacaine)

foot blood-flow and

an

tion,

motor

function, heart-rate, arterial pressure, and respira-

tory frequency. Sedation was moderate or absent. The potent and prolonged analgesia without central depreswe observed with intrathecal morphine accords with experimental data’ and with a preliminary report in man.2 The detectable thoracic upper level of analgesia we observed has not been previously reported in man and strongly supports a direct effect of narcotics, when given intrathecally, on the spinal cord. It would be interesting to know if Behar et al. have observed such a level of analgesia after epidural morphine. The delay of onset of analgesia we observed with 20 mg intrathecal morphine was longer than the delay recorded by Behar et al. with 2 mg epidural morphine (26 and 3 min, respectively). Such a rapid onset of analgesia may be due to the diffusion of morphine from the hypervascularised epidural

sion that

space into blood.

Nevertheless, we do agree with Behar et al. when they suggest that the unusual prolonged analgesia observed after a single administration of epidural morphine supports the hypothesis of a direct spinal action. K. SAMII J. FERET

Département d’Anesthésie-Réanimation, Hôpital Pitié-Salpêtrière, 75013 Paris, France

A. HARARI P. VIARS

SAFE EXPIRATORY VALVE FOR ANÆSTHESIA AND ARTIFICIAL VENTILATION

SIR,-Dr Wright (April 21, p. 854) offers anaesthetists the of simplicity. He expresses an enviable degree of confidence in his device when he describes it as "safe". When hand ventilation against high airway resistance is necessary, Wright offers two suggestion, one of which involves removing the valve from the system. I think I can accept the logic of his recommendation but I am opposed instinctively to the temporary removal of a safety device ("where did I put it?") when conditions are adverse. The alternative is to modify the valve to permit high-pressure ventilation by the finger (whose?). I find this suggestion even less acceptable because under difficult circumstances there is not usually a finger to

genius

-

Department of Anæsthesia and Intensive Care, School of Medicine, Flinders University of South Australia, Bedford Park, South Australia 5042

M. J. COUSINS L. E. MATHER C. J. GLYNN P. R. WILSON J. R. GRAHAM

SiR,—Dr Behar and colleagues report an analgesic effect of epidural morphine and suggest a direct spinal action of narcotic analgesics. We would like to report our experience of intrathecal morphine. Ten patients were given 20 mg morphine intrathecally as a hypertonic solution (morphine 1% and dextrose 15%). Immediately after the injection the patients were put in a 40 degree head-up position. Over a 48 h period intensity of pain (scored 0-5), level of analgesia by pinprick, fine sensation (epicritic sensibility), motor function, heart-rate, arterial pressure, respiratory frequency, and degree of sedation were recorded. Analgesia appeared at 26±4 min (mean±s.E.M.), reached a peak at 1 h, and disappeared at 27±2 h. The initial pain, which was severe (score 5), decreased to score 0 or 1 in all patients. The upper level of analgesia was easily detectable and ranged from Tl to T6. No change was observed in fine sensa8.

Cousins, M. J., and others. ibid. (in the press).

spare.

Wright’s modification of the standard Heidbrink valve is readily available, inexpensive, and effective. NeverthelessI should prefer a device that will permit ventilation at 6 kPa (just in excess of the standard 2 litre bag pressure limit) for short periods, the pressure automatically reverting to 3 kPa over several minutes after the higher pressure requirements have ceased. I have in mind a two-pressure setting device which could be "wound up" to the higher pressure and which would automatically "wind down" to the lower. If the need for high pressure persists the valve could be re-wound periodically, While the need for high-pressure ventilation is not an everyday occurrence it is not rare-perhaps, one case a month in busy general anaesthetic practice. Such cases are common in thoracic anaesthesia. Department of Anæsthesia,

Royal Free Hospital, London NW3 2QG

T. HILARY HOWELLS

FAST GLYCOSYLATION OF HÆMOGLOBIN

SIR,-Dr Welch (March 31, p. 728) and Dr Leslie and his colleagues (April 7, p. 773), commenting on our finding (March 17, p. 603) of fast glycosylation and de-glycosylation of haemoglobin in diabetics, maintain the view that the sessment

of

true, but

our

of

glycosylated haemoglobin is an aid to the aslong-term diabetic control. This might well be

measurement

critics

seem to

miss

our

point, which was that this

1. Yaksh, T. L., Rudy, T. A. Science, 1976, 192, 1352. 2. Wang, Y. K. Ann. Anesth. fr. 1978, 19, 371.

1143 has not yet been proved. Since there are considerable clinical and economic implications, the difference of opinion is not merely academic. Correct clinical use of any laboratory test requires that all possible problems in measuring and interpreting the variable in question be resolved. This applies to measurement of both HbA’e and HbA1. Rapid fluctuations in glycosylation of hxmoglobin could well be a pitfall of considerable importance in the assessment of diabetic control. Published work (and our own observations) does indeed demonstrate a correlation between diabetic control and HbAle or HbAI levels in groups of patients. However, to be useful to the clinician a good correlation must hold for individuals and for diabetics who are not in extreme states of very good or very bad regulation. To our knowledge, such a correlation has not been reported, and preliminary results from our clinic are not

tion of normal red blood-cells in a mixture of trace amounts of 14C-glucose and 40 mmol/1 unlabelled glucose. After subsequent incubation in glucose-free medium this radioactive peak disappeared. Furthermore, after incubation of normal red blood-cells in saline (37°C, 24 h) with trace amounts of 14C-glucose, radioactivity was eluted in the main HbA1e peak indicating that 14C-glucose was incorporated in h2emoglobin Ale although a net decrease was measured. This is further evidence that glucose in the HbA1e molecule is interchangeable with free glucose. Until methodological studies in depth and better clinical studies of the usefulness of HbA1e and HbA1 in assessment of long-term diabetic control in ordinary diabetic outpatients are available, scepticism is still justified. P. AABY SVENDSEN J. SANDAHL CHRISTIANSEN A. RENARD ANDERSEN B. WELINDER J. NERUP

convincing. Leslie et al. report on changes in HbAI during the first weeks of treatment in newly diagnosed diabetics. Decreases in HbAI of 5-24% (mean 15%) did occur during the first week. If we ignore the unlikely possibility that these patients had a considerable degree of haemolysis, this finding can be explained only by rather rapid intraerythrocytic deglycosylation. We are unable to confirm Welch’s report on the production of stable HbAI after incubation of normal red blood-cells in 100 mmol/1 glucose and subsequent washing in glucose-free medium and on the stability of HbAI in diabetic’s red bloodcells during washing in glucose-free saline. In red blood-cells from four controls the mean HbAle rose from 5.16% to 9.18% after 7 h incubation in 100 mmol/1 glucose in saline at 37°C but decreased to 5.4% after another 17 h incubation in glucosefree saline at 37°C. Thus only 6.2% of the rapidly formed HbAle remained in the cells. This is in contrast to 79-4% reported by Welch for HbAI. In our experiments changes in HbAla+b fraction were insignificant. Further, in blood from eight diabetics we found that the level of HbAle decreased from after incubation in 6 mean 9.74% to 8.81% (2p<0-001) volumes of glucose-free saline for 24 h at 37°C. No significant change was seen in the Ala+b fraction. The interesting suggestion that the chromatographically determined rapidly produced HbAle is a Schiff base adduct between glucose and haemoglobin cannot be answered in chemical terms, but as seen in the figure, the change in elution profile is small and of no practical importance because fast changes in HbA1e must be more than 10% and samples with and without "rapidly produced Ale" must be determined in the same series for the shift to be recognised; if HbA1 is collected in one fraction this shift in elution profile will not be detected. We have tried to elucidate further the process of fast glycosylation of haemoglobin, using 14 C-labelled glucose. A peak of radioactivity was found, coinciding with the ascending part of the left-shifted HbAle elution curve (see figure 1) after incuba-

Steno Memorial Hospital, DK-2820 Gentofte, Denmark

SIR,-Dr Svendsen and colleagues (March 17, p. 603) suggest that the formation of glycosylated haemoglobin (HbA,) is a fast and reversible process. To support this conclusion Svendsen et al. report the results of in-vitro and in-vivo experiments, suggesting that HbA1 levels are significantly affected by blood-glucose fluctuations over a few hours. Their in-vitro data, however, are in contrast with those of Dr Welch (March 31, p. 728). We find the in-vivo results of Svendsen et al.-a reduction to 10.42% after only 6 h of’Biostator’ treatment in a single poorly controlled diabetic patientvery surprising. Our preliminary findings on HbA1(a+b+e) levels before, during, and after three days of biostator treatment in eleven diabetics 1,2 showed that HbAI levels were stable for several days before the start of the study. Five insulin-treated diabetics were in fair metabolic control (group A), as expressed by

of HbA1e of from 12.07%

values (fasting 132+9.9 mg/dl; after lunch 130+13-8 mean+s.E.M.). The other six were diabetics on inap-

blood-glucose

treatment (group B), who showed poor metacontrol (blood-glucose 255±20.4 mg/dl fasting; 298±21-8after lunch). After a few hours of biostator treatment, the blood-glucose dropped to near-physiological levels in all patients and remained continuously controlled, not only during the fasting state (80-110 mg/dl) but also after meals (120-140 mg/dl). Daily blood volumes withdrawn for bloodglucose monitoring by the biostator was calculated at about 40 ml. Haematological indices (haematocrit, Hb, red-blood-cell count) were determined and reticulocytes were counted every 24 h of biostator treatment, and no significant changes were

propriate insulin bolic

1.

Bolli, G., Cartechini, M. G., Compagnucci, P., Cirotto, C., Massi-Benedetti, M., Santeusanio, F., Brunetti, P. I.R.C.S. med. Sci. 1979, 7, 30. 2. Brunetti, P., Massi-Benedetti, M., Puxeddu, A., Santeusanio, F., Calabrese, G., Bolli, G., Angeletti, G. Horm. Metab. Res. (in the press).

HbAI

LEVELS IN ELEVEN DIABETICS IN POOR

CONTROL OVER

I u

Change

LV

in elution pattern after

JV

EffLuent (mL) rapid (12 h)

3

YV u

increase in

HBA1eo

*

p<0’05

(B)

OR FAIR

DAYS ON AN ARTIFICIAL PANCREAS

(v.s. basal value) by analysis of variance.

(A)

1144 observed. HbAi(a+b+c) was determined by an improved chromatographic method.3.4 Estimation of HbA,(a+b+c) on duplicate samples was highly reproducible, 0-15% being the highest difference observed. HbAna+b+c) did not decrease in our patients 6 and 12 h after biostator treatment. The results observed after 24, 48, and 72 h are summarised in the table (mean values of duplicate determinations at each sampling were measured). Our data clearly show that the decrease of HbA1 during biostator treatment is very slight and becomes statistically significant only after 72 h of feedback insulin infusion. For a careful investigation of the effects of biostator control on HbA1 only diabetics with normal and/or constant redblood-cell half-life should be considered. In our patients hsematological indices and reticulocyte-counts were normal and did not vary

before, during, and after biostator treatment.

We have shown that HbA1 levels may fall significantly within a few days during strict glucose metabolic control achieved for diabetics by the biostator. Variations of HbA

be observed within a few hours, as reported al. by HbA1 determination, therefore, provides the diabetologist with information on metabolic control over the previous weeks and also, at least in part, over the previous few days. GEREMIA BOLLI MARIA G. CARTECHINI PIETRO COMPAGNUCCI Institutes of Medical Pathology MASSIMO MASSI-BENEDETTI and Clinical Medicine, FAUSTO SANTEUSANIO University of Perugia, PAOLO BRUNETTI Perugia, Italy

however,

cannot

Svendsen

et

IMPROVED FLUORESCENT STAINING OF INTERPHASE NUCLEI FOR PRENATAL DIAGNOSIS

SIR,-The prenatal diagnosis of chronic granulomatous disease

of childhood’ has been

accomplished by

a

slide

test

for

3. Cirotto, C., Panara, F., Compagnucci, P., Cartechini, M. G., Bolli, G., Santeusamo, F. I.R.C.S. med. Sci. 1978, 6, 374. 4. Cirotto, C., Arangi, I., Compagnucci, P., Cartechini, M. G., Bolli, G., Nicoletti, I.ibid. 1978, 6, 375. 1. Newburger, P. E., Cohen, H. J., Rothchild, S. B., Hobbins, J. C., Malawista, S. E., Mahoney, M. J. New Engl. J. Med. 1979, 300, 178.

nitroblue tetrazolium (N.B.T.) reduction by human fetal phagocytes (granulocytes and monocytes). The disease has a 7:1 case ratio of X-linked to autosomal recessive -inheritance; thus most fetuses at risk are male. Since blood-samples obtained by fetoscopic placental-vessel puncture may be contaminated with maternal blood,3 we used quinacrine hydrochloride as a counterstain to identify male (i.e., fetal) cells by Y-chromatin fluorescence. However, the N.B.T. reaction appeared to quench quinacrine fluorescence, resulting in decreased differentiation of the Y bodies and delicate requirements for incubationtimes.t To improve the quality of fluorescent staining for Y chromatin in the N.B.T. slide test, we sought to apply previous work with intermolecular energy transfer between fluorescent dyes4,5 to enhance contrast. Quinacrine-bright regions, such as the human Y body, resist quenching by energy transfer to dyes with G-C binding specificity, such as 7-aminoactinomycin D (7-A.A.),6 which diminish quinacrine fluorescence from most other chromosomal regions. To test the method, we obtained leucocytes from dextransedimented blood of a male donor known to have no large quinacrine-bright autosomal polymorphisms. Both plain smears and phorbol-myristate-acetate stimulated N.B.T. slides (prepared as previously described’) were stained with quinacrine hydrochloride (20 mg/ml) for 5 min, then rinsed sequentially in water, McIlvaine’s citrate-phosphate buffer (pH 5’!) for 40 s, and water again. Nuclei were observed either directly or after counterstaining with 7-A.A. (5 rrtol/1) in 1:5 diluted Mcllvaine buffer. B As shown in the accompanying figure, an N.B.T. slide stained with quinacrine (panel A) revealed Y bodies in granulocyte nuclei, but they are indistinct due to quenching by the reduced N.B.T. In contrast, 7-A.A.-enhanced Y body fluorescence (panel B) was resistant to quenching. Y-body differentiation in samples viewed directly by fluorescence microscopy actually exceeds that in the black and white photograph shown, since the Y body appears bright yellow-green against a dull red background provided by 7-A.A. fluorescence. This finding represents a major improvement over previous experience with quinacrine alone, in which quenching of fluorescence by the N.B.T. reaction decreased detection of Y bodies and hence of male cells. Thus the enhancement of contrast in fluorescence from quinacrine by energy transfer to 7-A.A., previously described for banding patterns of metaphase chromosomes,4,s provides a means for improved detection of Y-chromatin bodies in interphase nuclei of human phagocytes. The dye pair also protects the detectability of the Y body from deterioration after phorbolmyristate-acetate induced N.B.T. reduction, which quenches the fluorescence of quinacrine alone. Double fluorescent staining to detect Y-chromatin bodies using quinacrine and 7-A.A. or actinomycin D6 appears to be a superior method for the identification of male fetal cells-e.g., for individual evaluation of N.B.T. reduction in the prenatal diagnosis of chronic granulomatous disease or for other potential applications relating to prenatal diagnosis in which sex determination is based on fluorescent staining of interphase nuclei. We thank Ms, Lois Jurgens for expert technical assistance and the National Institutes of Health and the National Foundation-March of Dimes for financial suooort. Divisions of Hematology/Oncology and Children’s Hospital Medical Center, and Division of Genetics, Boston Hospital for Women, Boston, Massachusetts 02115, U.S.A.

Fluorescence nuclei.

photomicrographs

of human

leucocyte interphase

N.B.T. slides of cells from a single donor were processed simultaneously and stamed with quinacrme (panel A or quinacrine plus 7-A.A. (panel B). Arrows indicate Y bodies. (x675.)

2. 3. 4. 5.

Genetics,

PETER E. NEWBURGER SAMUEL A. LATT

Johnston, R. B., Jr., Baehner, R. L. Pediatrics, 1971, 48, 730. Alter, B. P., Nathan, D. G. Clins Hœmat. 1978, 7, 195. Sahar, E., Latt, S. A. Proc. natn. Acad. Sci. U. S. A. 1978, 75, 5650. Latt, S. A., Sahar, E., Eisenhard, M. E. J. Histochem. Cytochem. 1979, 27,

65. 6. Modest, E. J., Sengupta,

S. K. Cancer Chemother. Rep. 1974, 58, 35.